Integrated high throughput system for the analysis of biomolecules

ABSTRACT

Described is an affinity microcolumn comprising a high surface area material, which has high flow properties and a low dead volume, contained within a housing and having affinity reagents bound to the surface of the high surface area material that are either activated or activatable. The affinity reagents bound to the surface of the affinity microcolumn further comprise affinity receptors for the integration into high throughput analysis of biomolecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/035,942 and entitled “Integrated High Throughput System for theAnalysis of Biomolecules” filed on Feb. 26, 2011, currently pending,which is a continuation of U.S. patent application Ser. No. 12/643,798and entitled “Integrated High Throughput System for the Analysis ofBiomolecules” filed on Dec. 21, 2009, now abandoned, which is acontinuation of U.S. patent application Ser. No. 11/843,826 and entitled“Integrated High Throughput System for the Analysis of Biomolecules”filed on Aug. 23, 2007, now U.S. Pat. No. 7,635,425, which is adivisional of U.S. patent application Ser. No. 11/462,933 and entitled“Integrated High Throughput System for the Analysis of Biomolecules”filed on Aug. 7, 2006, now U.S. Pat. No. 7,311,826, which is acontinuation of U.S. patent application Ser. No. 10/710,994 and entitled“Integrated High Throughput System for the Analysis of Biomolecules”filed on Aug. 16, 2004, now U.S. Pat. No. 7,087,165, which is adivisional of U.S. patent application Ser. No. 10/053,098 and entitled“Integrated High Throughput System for the Mass Spectrometry ofBiomolecules” filed on Jan. 15, 2002, now U.S. Pat. No. 6,783,672, whichis a continuation-in-part of provisional applications 60/262,530 and60/262,852, both filed on Jan. 18, 2001.

FIELD OF INVENTION

The present invention is related to the field of proteomics. Morespecifically, the present invention is a method and device for rapididentification and characterization of biomolecules recovered frombiological media. Additionally, the present invention includes theability to process numerous different samples simultaneously (highthroughput analysis).

BACKGROUND OF THE INVENTION

Recent advances in human genome sequencing have propelled the biologicalsciences into several new and exciting arenas of investigation. One ofthese arenas, proteomics, is largely viewed as the next wave ofconcerted, worldwide biological research. Proteomics is theinvestigation of gene products (proteins), their various different formsand interacting partners and the dynamics (time) of their regulation andprocessing. In short, proteomics is the study of proteins as theyfunction in their native environment with the overall intention ofgaining a further, if not complete, understanding of their biologicalfunction. Such studies are essential in understanding such things as themechanisms behind genetic disorders or the influences of drug mediatedtherapies, as well as potentially becoming the underlying foundation forfurther clinical and diagnostic analyses.

There are several challenges intrinsic to the analysis of proteins.First, and foremost, any protein considered relevant enough to beanalyzed resides in vivo in a complex biological environment or media.The complexity of these biological media present a challenge in that,oftentimes, a protein of interest is present in the media at relativelylow levels and is essentially masked from analysis by a large abundanceof other biomolecules, e.g., proteins, nucleic acids, carbohydrates,lipids and the like. Technologies currently employed in proteomics areonly able to overcome this fundamental problem by first fractionatingthe entire biological media using the relatively old technology oftwo-dimensional (2D) sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE), wherein numerous proteins are simultaneouslymigrated using a gel medium, in two dimensions as a function ofisoelectric point and molecular size. In order to ensure migration in apredictable manner, the proteins are first reduced and denatured, aprocess that destroys the overall structures of the proteins and voidstheir functionality.

Present day state-of-the-art proteomics involves the identification ofthe proteins separated using 2D-PAGE. In this process, gel spotscontaining separated proteins are excised from the gel medium andtreated with a high-specificity enzyme (most commonly trypsin) tofragment the proteins. The resulting fragments are then subjected tohigh-accuracy mass analysis using either electrospray ionization (ESI)or matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometries (MS). The resulting data, in the form ofabsolute molecular weights of the fragments, and knowledge of the enzymespecificity are used in silico to search genomic or protein databasesfor information correlating to the empirical data on the fragments.Analytical methods and searching protocols, refined over the past sevenyears, have evolved to a point where only a few proteolytic fragments,determined with high mass accuracy, are needed to identify agel-separated protein as being present in a certain gene.

However, identification of the gene producing a protein of interest isonly the first step in the overall, much larger process of determiningprotein structure/functionality. Numerous questions that arise cannot beanswered by the 2D-PAGE/MS approach. One major issue deals with theprimary structure of the protein. During the commonly practicedidentification process, at most, fifty percent of the protein sequenceis viewed, leaving at least fifty percent of the protein unanalyzed.Given that potentially numerous splice variants, point mutations, andpost-translational modifications exist for any given protein, manyvariants and modifications present within a protein will ultimately bemissed during the identification process—many of which are responsiblefor disease states. As such, proteins are not viewed in the fullstructural detail needed to differentiate (normal) functional variantsform (disease-causing) dysfunctional variants.

Furthermore, current identification processes make no provision forprotein quantitation. Because many disease states are created orindicated by elevated or decreased levels of specific proteins and/ortheir variants, protein quantitation is a very important component ofproteomics. Presently, protein quantitation from gels is performed usingstaining approaches that inherently have a relatively high degree ofvariability, and thus inaccuracy. The staining approaches can bereplaced using isotope-coded affinity tags (ICAT) in conjunction withmass spectrometric quantification of proteolytic fragments generatedfrom 2D-PAGE. However, the ICAT approach is still subjective to theaforementioned protein variants in that protein variants will yieldmass-shifted proteolytic fragments that will not be included in thequantification process. Likewise, other approaches, such as ELISA(enzyme-linked immunosorbant assay) and RIA (radioimmunoassay), areequally subjected to the complications of quantifying a specific proteinin the presence of its variants. Lacking the ability to resolve a targetprotein from its variants, these techniques will essentially monitor allprotein variants as a single compound; a process that is oftentimesmisleading in that a disease may be caused/indicated by elevated levelof only a single variant, not the cumulative level of all the variants.

Moreover, the 2D-PAGE/MS approaches make no provision for exploringprotein-ligand (e.g., other proteins, nucleic acids or compounds ofbiological relevance) interactions. Because denaturing conditions areused during protein separation, all protein-ligand interactions aredisrupted, and thus are out of the realm of investigation using theidentification approach. Separate other approaches focus specifically onthe analysis of protein-ligand interactions. The most frequently used ofthese are the yeast two-hybrid (Y2H) and phage display approaches, whichuse in vivo molecular recognition events to trigger the expression ofgenes that produce reporter proteins indicating a biomolecularinteraction, or selectively amplify high-affinity binding partners,respectively. Other instrumental approaches rely on biosensors utilizinguniversal physical properties or tags (e.g., surface plasmon resonanceor fluorescence) as modes of detection. The two major limitations ofthese approaches is that they are generally slow and that interactingpartners pulled from biological media are detected indirectly, yieldingno specific or identifying information about the binding partner.

Lastly, none of the aforementioned approaches are favorable tolarge-scale, high-throughput analysis of specific proteins, theirvariants and their interacting partners in large populations ofsubjects. All of the aforementioned approaches require several hours(2D-PAGE) to several weeks (Y2H) to perform on a single sample. As such,time and, monetary expenses preclude application to thehundreds-to-thousands of samples (originating from hundreds-to-thousandsof individuals) necessary in proteomic, clinical, and diagnosticapplications.

To date, there are no universal, integrated systems capable of thehigh-throughput analysis of proteins for all of the aforementionedreasons. Thus, there exists a pressing need for new and noveltechnologies able to analyze native proteins present in their naturalenvironment. Encompassed in these technologies are: 1) the ability toselectively retrieve and concentrate specific proteins from biologicalmedia for subsequent high-performance analyses, 2) the ability toquantify targeted proteins, 3) the ability to recognize variants oftargeted proteins (e.g., splice variants, point mutations andposttranslational modifications) and to elucidate their nature, 4) thecapability to analyze for, and identify, ligands interacting withtargeted proteins, and, 5) the potential for high-throughput screeningof large populations of samples using a single, economical platform.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. Although the present invention has beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it will be apparent that certain changesand modifications may be practiced within the scope of the appendedclaims.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an integrated systemcapable of selectively retrieving and concentrating specificbiomolecules from biological media for subsequent high-performanceanalyses, quantifying targeted proteins, recognizing variants oftargeted biomolecules (e.g., splice variants, point mutations andpost-translational modifications) and elucidating their nature,analyzing for, and identifying, ligands interacting with targetedbiomolecules, and high-throughput screening of large populations ofsamples using a single, unified, economical, multiplexed and parallelprocessing platform.

It is another embodiment of the present invention to provide anintegrated system that comprises molecular traps, such as affinitymicrocolumns, derivatized mass spectrometer targets, mass spectrometerscapable of multi-sample input and robotics with processing/data analysisinteractive database software that accomplish the high throughputanalysis.

It is yet another object of the present invention to provide individualcomponents for the integrated system, such as molecular traps,derivatized targets and the like.

It is a further object of the present invention to provide a highthroughput embodiment of the present invention that uses robotics forserial preparation and parallel processing of a large number of samplessimultaneously.

It is yet a further object of the present invention to provide methodsand processes for use of the individual components and the integratedsystem in biological applications.

It is still yet another object of the present invention to provide adevice and method for the identification of point mutations and variantsof analytes using an integrated system using high throughput analysis.

The novel features that are considered characteristic of the inventionare set forth with particularity in the appended claims. The inventionitself, however, both as to its structure and its operation togetherwith the additional objects and advantages thereof will best beunderstood from the following description of the preferred embodiment ofthe present invention when read in conjunction with the accompanyingdrawings. Unless specifically noted, it is intended that the words andphrases in the specification and claims be given the ordinary andaccustomed meaning to those of ordinary skill in the applicable art orarts. If any other meaning is intended, the specification willspecifically state that a special meaning is being applied to a word orphrase. Likewise, the use of the words “function” or “means” in theDescription of Preferred Embodiments is not intended to indicate adesire to invoke the special provision of 35 U.S.C. §112, paragraph 6 todefine the invention. To the contrary, if the provisions of 35 U.S.C.§112, paragraph 6, are sought to be invoked to define the invention(s),the claims will specifically state the phrases “means for” or “step for”and a function, without also reciting in such phrases any structure,material, or act in support of the function. Even when the claims recitea “means for” or “step for” performing a function, if they also reciteany structure, material or acts in support of that means of step, thenthe intention is not to invoke the provisions of 35 U.S.C. §112,paragraph 6. Moreover, even if the provisions of 35 U.S.C. §112,paragraph 6, are invoked to define the inventions, it is intended thatthe inventions not be limited only to the specific structure, materialor acts that are described in the preferred embodiments, but inaddition, include any and all structures, materials or acts that performthe claimed function, along with any and all known or later-developedequivalent structures, materials or acts for performing the claimedfunction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the MSIA procedure. Analytes areselectively retrieved from solution by repetitive flow through areceptor-derivatized affinity pipette. Once washed of thenon-specifically bound compounds, the retained species are eluted onto amass spectrometer target or target array using a MALDI matrix (in thepreferred embodiment). MALDI-TOF MS then follows, with analytes detectedat precise m/z values. The analyses are qualitative by nature but can bemade quantitative by incorporating mass-shifted variants of the analyteinto the procedure for use as internal standards.

FIG. 2 β₂-microglobulin MSIA screening of biological fluids. Sampleswere prepared by dilution of the biological fluid with HBS (H₂O forstandalone MALDI-TOF) and repetitive flow incubation through theaffinity pipette. Affinity pipettes were washed using HBS and waterbefore elution of retained compounds directly onto a mass spectrometertarget using ACCA (saturated in 1:2, ACN:H₂O; 0.2% TFA). (A) Humantears. (B) Human plasma. (C) Human saliva the saliva required anadditional rinse with 0.05% SDS (in water) to reduce non-specificbinding. (D) Human urine. In all cases, β₂m was efficiently retrievedfrom the biological fluids using the flow-incubate/rinse procedure. Themasses determined for the β₂m (using external calibration) were within˜0.1% of the calculated value (MW_(calc)=11,729.7; MW_(tears)=11,735;MW_(plasma)=11734; MW_(saliva)=1 1,742; MW_(urine)=11,735). Illustratingdiverse biological fluid screening by MSIA for a directed, rapid,sensitive and accurate analysis.

FIG. 3 a Quantitative β₂m-MSIA—working curve. Representative spectra ofdata used to generate the working curve. Human β₂m concentrations of0.01-1.0 mg/L were investigated. Equine β₂m (MW=11,396.6) was used as aninternal standard.

FIG. 3 b Working curve generated using the data represented in FIG. 3 a.The two-decade range was spanned with good linearity (R²=0.983) and lowstandard error (˜5%). Error bars reflect the standard deviation oftenrepetitive 65-laser shots spectra taken from each sample. These figuresillustrate quantitative MSIA performed via β₂m-affinity pipettes.

FIG. 4 Quantitative β₂m-MSIA—screening. Human urine samples from fiveindividuals were screened over a period of two days. The average valuedetermined for healthy individuals (10-samples; 4-individuals (3 male; 1female) ages 30-44 years) was 0.100±0.021 mg/L. The level determined foran 86-year old female with a recent urinary tract infection indicated asignificant increase in β₂m concentration (3.23±0.072 mg/L).

FIG. 5 MSIA showing elevated level of glycosylat ed β₂m in a 86-year oldfemale (dark gray). During MSIA, a second signal is observed at Δm=+161Da, indicating the presence of glycosylated β₂m. MSIA is able toadequately resolve the two β₂m forms, resulting in a more accuratequantification of the nascent β₂m and possible quantification of theglycoprotein. Such differentiation is important considering that the twoβ₂m forms originate from (or are markers for) different ailments. MSIAof a healthy individual, showing little glycosylation, is given forcomparison (light gray).

FIG. 6 Surface directed MSIA for defined biologicalfluid/β₂-microglobulin specificity. Use of polyclonal anti-β₂m affinitypipettes linked via carboxymethyl dextran amplification or amine basesupport chemistries enable differentiation of specifically bound versusnon-specifically bound compounds during biological fluid/MSIA. Sampleswere prepared from biological fluid and used as in FIG. 2. (A) Humanplasma. (B) Human plasma through CMD amplified β₂m affinity pipette. (C)Human plasma through amine/glutaraldehyde coupled β₂m affinity pipette.Direct analysis of human plasma spectra (top spectrum) lacks β₂m masssignature (MW_(plasma)=11734). CMD amplified affinity pipette chemistrytarget β₂m while exhibiting non-specifically bound compounds (middlespectrum). Only in the last case was β₂m efficiently retrieved from thebiological fluid with low non-specifically bound compounds (bottomspectrum). This illustrates a preferred surface in the directed analysisof blood born biological fluid biomarkers using discreet affinitypipettes for specific mass detection.

FIG. 7 a is a schematic illustration describing the integrated systemfor high-throughput analysis of biomolecules from biological media.

FIG. 7 b is an expanded schematic illustration of the used station,which is comprised of microcolumn-integrated robotics having multiplepositions for chemical modification, microcolumn functionalization,biological fluids analysis, transfer and the like.

FIG. 8 is an illustration of a high-throughput semi-quantitativeanalysis of β₂m MSIA. β₂m from human plasma samples using the integratedsystem and methods described in the present invention.

FIG. 9 shows bar graph analysis of the data shown in FIG. 8. Eachspectrum shown in FIG. 8 was normalized to the equine β₂m signal throughbaseline integration, and the normalized integral for the human β₂msignal determined. All β₂m integrals from spectra obtained from samplefrom the same individual were averaged and the standard deviationcalculated. In the same way, the integrals for the samples spiked with0.5 and 1.0 solution of 10⁻² mg/mL β₂m were calculated and averaged.Plotted in this figure are the average values of the normalized humanβ₂m integrals for the samples from the six individuals and the spikedsamples. The bar graph clearly establishes increased β₂m levels in thespiked samples, illustrating the value of the high-throughputsemi-quantitative analysis performed with the system and methodsdescribed in this invention in establishing increased β₂m levels inhuman blood that are associated with various disease states.

FIG. 10 is an illustration of a high-throughput quantitative analysis ofβ₂m from human plasma samples using the integrated system and methodsdescribed in this invention.

FIG. 11 a and 11 b illustrate the construction of a calibration curvefrom the data for the standard samples shown in FIG. 10 and for thepurpose of determining the β₂m concentrations in the human plasmasamples screened via the high-throughput analysis using the integratedsystem and methods described in this invention.

FIG. 12 shows bar analysis of the data shown above using the standardcurve constructed above. Each spectrum for the 88 samples in FIG. 10 wasnormalized to the equine β₂m signal through baseline integration, andthe normalized integral for the human β₂m signal determined. All humanβ₂m integrals for the same individual were averaged and the standarddeviation calculated. The values of the averaged integrals weresubstituted in the equation derived from the standard curve, FIG. 11 b,and the concentration of human β₂m was calculated for each individual.The range of concentrations determined was from 0.75 to 1.25 mg/L.

FIG. 13 is an illustration of a qualitative high-throughput screening oftransthyretin (TTR) for posttranslational modification (PTM) and pointmutations (PM) using the integrated system and methods described in thisinvention.

FIG. 14 illustrates identification of the posttranslationalmodifications and point mutations observed in the high-throughput TTRanalysis using the integrated system and methods described in thisinvention.

FIG. 15 illustrates the identification of point mutation viaincorporation of derivatized mass spectrometer target platforms in thesystem and methods described in this invention.

FIG. 16 illustrates the use of a high-resolution reflectron massspectrometry as part of the integrated system and methods described inthis invention in determining the identity of the point mutationsdetected in the analysis of the plasma samples shown in FIG. 15.

FIG. 17 a Concerted biofluid phosphate analysis-chelator affinitypipettes with alkaline phosphatase functionalized target array. (1)Human whole saliva (10 μL diluted 10 fold), (2) sample in (1) throughEDTA/Ca²⁺ affinity pipettes, (3) sample in (2) eluted via 10 mM HCladdition and stamped onto AP-BRP incorporating 50 mM borate buffer pH-10buffer exchange and fifteen minute phosphate digest (50° C.), (4) samplein (3) with extended thirty minute digest. Direct analysis of ten bydilution of human saliva significantly lacks proline rich protein-1(PRP-1), the serine modified phosphate rich protein of interest.

FIG. 17 b Spectrum in (2) shows EDTA/Ca²⁺ affinity pipette capture oftwo phosphate rich proteins, PRP-1 and PRP-3. Mass signature ofdephosphorylation is evident in spectral trace (3) and complete in (4).Illustrating multi-analyte detection accompanied by partial and completedephosphorylation of phospho-proteins captured/digested out ofbiological fluid for post-translational analysis (i.e., phosphorylationevents.

FIG. 18 MSIA delineation of multi-protein complex between retinolbinding protein (RBP) and transthyretin (TTR). Polyclonal anti-RBPaffinity pipettes were formed via glutaraldehyde mediated amine basesupport surface coupling. Human plasma was prepared and used as in FIG.8. MSIA shows in vivo affinity retrieval of RBP (MW=21,062 Da) andcomplexed TTR (MW=13,760 Da). Illustrating protein interactions exitingin native protein complexes.

FIG. 19 Simultaneous rapid monitoring of multi-analytes for relativeabundance. Amine activated, polyclonal anti-β₂m/CysC/TTR affinitypipettes are used to rapidly capture their respective analytes out ofhuman plasma (50 fold diluted in HBS). The figure illustrates one of theuses for multi-antibody affinity pipettes to β₂m, CysC and TTR torapidly monitor for biological fluid level modulation and to quantify amodulated protein event from their normalized relative abundance. Thefigure illustrates one of the uses of affinity pipettes for monitoringpotential β₂m/CysC levels in acute phase of viral infection (ca. AIDS)or fibril formation from β₂m or TTR.

FIG. 20 Rapid monitoring of extended multi-analyte affinity pipettes.Combinations/individual polyclonal antibody affinity pipettesincorporating β₂m, TTR, RBP, Cystatin C or CRP capture respectiveanalytes from human plasma (50 fold dilution in HBS). This figureillustrates one of the uses for multi/single-antibody affinity pipettesto β₂m, CysC, TTR or CRP to rapidly monitor for biological fluid levelmodulation and to potentially quantify a modulated protein event fromtheir normalized relative abundance. This figure illustrates another ofthe uses of affinity pipettes for monitoring potential β₂m/CysC levelsin acute phase of viral infection (ca. AIDS) or fibril formation fromβ₂m or TTR.

FIG. 21 Mass spectrometry target arrays. (A) Plateau target capable ofconfining sample through meniscus action. (B) Contrast design capable ofconfining sample through hydrophobic/hydrophilic action. (C) Inserttargets for use with smaller sampling loads, expensive reagents, orsample transfers.

DETAILED DESCRIPTION

The present invention provides an integrated high throughput systemcapable of selectively retrieving and concentrating specificbiomolecules from biological media for subsequent high-performanceanalyses, such as identification of biomolecules, quantifying targetedbiomolecules, recognizing variants of targeted biomolecules (e.g.,splice variants, point mutations and post-translational modifications)and elucidating their nature, such as analyzing for, and identifying,ligands interacting with the targeted biomolecules, and high-throughputscreening of large populations of samples using a single, unified,economical, multiplexed and parallel processing platform.

The preferred embodiment of the integrated system comprises moleculartraps, such as affinity microcolumns, processing stations, andderivatized mass spectrometer target arrays, which may be omitted innon-preferred embodiments, that work with mass spectrometers capable ofsingle or multi-sample input and using processing/data analysisinteractive databases. The present invention also includes methods andprocesses for use of the individual components and the integrated systemin biological applications. Furthermore, the preferred embodiment of thepresent invention provides for the preparation and/or processing ofmultiple separate devices and/or samples to accomplish high throughputanalysis.

A major component of the system of the present invention is theisolation or retrieval of specific analytes from their surroundingbiological media in a biological sample. This is accomplished using amolecular trap. In a preferred embodiment of the molecular trap, theretrieval process entails repetitively flowing the biological samplethrough devices that have affinity receptors located on surfaces with ahigh surface area content. The affinity receptors are selected tocapture specific analytes. In the high throughput embodiment, thesemolecular traps are formed into miniature columns, affinitymicrocolumns, thereby allowing numerous molecular traps to be locatedside-by-side and taking up minimal amount of physical volume. In apreferred form of the side-by-side embodiment, the numerous moleculartraps are contained within a unitary component, such as a manifold orblock of material. In this form the manifold contains numerousmicrochannels that house the molecular traps.

The molecular trapping process is accomplished by allowing sufficientphysical contact between the affinity receptors located on the moleculartraps and the analyte contained in the biological sample. The affinityreceptors capture, or isolate, the specific analytes using an affinityinteraction between the affinity receptors and the specific analytes.After the specific analytes are captured, residual or non-capturedcompounds are washed free of the molecular traps using a series ofrinses. The capture and rinse processes result in the concentrating ofthe specific analytes into the low dead-volume of the affinitymicrocolumns.

After the specific analytes have been captured, they are eluted from themolecular traps using a small volume of a reagent capable of disruptingthe affinity interaction. The eluted specific analytes are then stampeddirectly onto a mass spectrometry target platform for either massspectrometry or for further processing, e.g., enzymatic/chemicalmodification via utilization of bioreactive MS target arrays, followedby subsequent preparation for mass spectrometry. Automated massspectrometry then follows with either the specific analyte or modifiedfragments detected with high precision. Software capable of recognizingdifferences between samples, or from a standard, is used to aid in theanalysis and organization into database of the large numbers of samples.Alternately, instead of stamping the eluted specific analytes onto amass spectrometry target, the specific analytes may be eluted directlyinto an electrospray ionization mass spectrometer by using the moleculartraps as a component in the sample introduction device, such as theneedle of an electrospray mass spectrometer.

The high throughput embodiment of the present invention uses roboticsfor serial preparation and parallel processing of a large number ofsamples. The use of microcolumns in capturing the specific analytesenables an arrayed format, as mentioned above, that is ideal for suchhigh-throughput processing since it minimizes the physical volume and/orarea occupied by the microcolumn array. Use of affinity microcolumnswith appropriately configured robotics allows multiple samples to beprepared, processed, start-to-finish, simultaneously on a unifiedplatform thereby enabling high throughput of samples. Specifically, allcapture, separation and elution steps are performed within themicrocolumns managed by the robotics system or systems. This is incontrast to the use of other affinity capture methods (using, e.g.,beaded media) where mechanical/physical means (e.g., centrifugation,magnetic or vacuum separation) are used to separate the specific analytefrom the biological fluid and rinse buffers. Oftentimes this physicalseparation needs to be performed singularly, resulting in the disruptionof a parallel processing sequence, as well as the ordering of the array.Because these mechanical/physical means are not necessary when using themicrocolumns, parallel-processing sequences can be used withoutdisruption and the integrity of an ordered spatial array is maintainedthroughout the entire process. Most conveniently, multiplepreparations/analyses are performed serially and in parallel usingrobotics fitted to commonly used spatial arrays, e.g., 4-, 8-, 16-, 48-,96-, 384 or 1536 well micro-titer plate formats.

Individual Components Sample Modification/Preparation

In all of the below described embodiments, it may be desired that thebiological media or the target analyte be modified or prepared eitherprior to affinity action or after affinity capture, but before elutiononto a target or target array. Example modifications or preparationsinclude, but are not limited to, reduction, labeling or tagging, in situdigests, partial on-surface digestion/modification, pH adjustments, andthe like.

Molecular Traps

In one embodiment of the invention, molecular traps are microcolumnardevices that have bound affinity receptors. The molecular trap ischemically modified, such as by treatment with an amino-silanizationreagent and subsequently activated for affinity receptor linkage usingany one of a number of derivatization schemes. The use of affinitymicrocolumns overcomes the disadvantages entailed in performing affinitycapture by other means. Specifically, affinity microcolumns, asdescribed herein, are scaled to mass spectrometric analyses that haveonly become available in the last ten years. Prior to the advent ofMALDI-TOF and ESI mass spectrometries, mass spectrometric analyses ofpolypeptides (if they could be performed at all) required amounts ofanalyte on the order of nanomoles, which, if isolated via affinitycapture, required milliliter volumes of reagent containing boundreceptors and, oftentimes, liter volumes of biological media. Given thelow-to sub-femtomole sensitivities of MALDI-TOF and ESI massspectrometries, the entire affinity isolation processes, includingdevices, can be scaled down by several orders of magnitude. Therefore,the affinity microcolumns described herein are devised and manufacturedto fully utilize the sensitivity specifications of the recent enablingmass spectrometric techniques.

An additional embodiment of the present invention is to provide avariety of affinity microcolumns specifically tailored to excel in agiven biological media, illustrated in FIG. 6. Because all biologicalmedia are not exactly the same, with regard to biomolecule compositionsand conditions, each affinity reagent derivatization scheme will behavedifferently in each biological media. For instance, affinity reagentstailored to retrieve a specific protein analyte present in plasma maynot behave ideally when targeting the same analyte when present in adifferent biological media. Furthermore, the different buffercompositions and conditions of each biological media make availablenumerous small organic compounds that when retained and subsequentlyeluted with the targeted analyte will potentially deter from the massspectrometric process. It is therefore necessary to construct affinitymicrocolumns for each biological fluid that show not only highspecificity towards targeted analytes and low non-specific bindingproperties with regard to other large molecules that potentiallyinterfere with the characterization of the analyte, but also exhibitminimal retention of smaller molecules that potentially interfere withthe physical phenomena underlying mass spectrometric processes, e.g.,MALDI or ESI.

Targets

After analytes are retrieved from biological media they are essentiallymicroeluted and “stamped” directly from the affinity microcolumns onto atarget or target array fitting into a mass spectrometer. In this manner,the spatial array from the initial multi-sample container, e.g., titerplate, is maintained throughout the affinity capture and washing steps,as well as onto the mass spectrometer target.

The present invention further embodies the use of specially tailoredmass spectrometer targets in the automated preparation and analysis ofproteins retrieved using the affinity microcolumns. Essential toincorporating the automated robotics into the high throughput, parallelprocess is reproducibility between each sample, and the ability tocontrol the location of the samples upon deposition onto the massspectrometer target. In order to ensure these aspects are instilled intothe automated process, self-assembled monolayers (SAM) are patternedonto mass spectrometer targets in manners able to control the area ofanalyte deposition. For example, thiol or mercaptan compounds that arehydrophobic or hydrophillic in character are used to pattern contrastingareas on gold-plated targets. By surrounding a hydrophillic SAM with ahydrophobic SAM, a clear boundary is created that is able to confineaqueous sample (from the affinity microcolumns) to a clearly definedarea on the target. The spatial array dictated by the parallel roboticscan thus be maintained by simultaneously eluting multiple samples, frommultiple affinity microcolumns (using robotics), onto a massspectrometer target patterned to the same spatial array used inthroughout the robotic processing.

In other applications, mass spectrometer targets are additionallytailored to include reactive surfaces capable of analyte processing.When investigating biomolecules using mass spectrometry it is oftennecessary to perform telltale chemistries and/or enzymologies to gainfurther detail on the structure of an analyte. Of particular importanceare analyses that use specific chemical or enzymatic modifications incombination with mass spectrometry for purposes such as identifyinganalytes, analyte variants and modifications present within an analyte.Moreover, oftentimes it is of great value to quasi-purify massspectrometric preparations by removal of potentially interfering speciesfrom solution through scavenging interactions designed to remove theinterferences while leaving the target analyte available of analysis. Amost efficient means of performing these operations is to use massspectrometer targets that are derivatized with chemicals or enzymes forthe particular processing function.

In a preferred embodiment, a target or target array is made by firstetching channels around designated target areas using photoresisttechnologies. A layer of gold is deposited onto the etched substrate,such as by traditional electro-plating techniques or plasma deposition.This layer of gold naturally follows the surface contours created by theetching. Depending upon the substrate, one or more intermediate layersmay be required, such as a nickel intermediate layer is required whendepositing a surface layer of gold. An activated or activatable reagent,capable of forming a chemical bond or adsorbing to the modifiedsubstrate surface, such as dithiobis(succinimidylproprionate) (DSP) orderivatives thereof, is then bound onto the target areas, but notelsewhere. Any transport solvent is either allowed to evaporate orremoved producing a dry self-assembled monolayer (SAM) of the activatedor activatable reagent. A protective layer is deposited onto the targetSAM, such as dextran solubilized in appropriate solvent. When thatsolvent is DMSO, the DMSO is then removed by placing the target in avacuum. The target (array) is then coated with a hydrophobic reagent,such as octadecyl mercaptan solubilized in a solvent that does notdissolve the protective layer. When dextran is the protective layer,isopropanol may be used to solubilize the hydrophobic reagent. Thetarget is rinsed to remove any non-bound hydrophobic reagent. In theexample where activated reagent is bound to the target areas, theactivated reagent is made available for use by merely removing theprotective layer, which also removes any hydrophobic reagent present inor on the protective layer, such as by rinsing with DMSO. In the examplewhere activatable reagent is bound to the target areas, the reagent maybe activated for use by either removing the protective layer followed byreagent activation using an activating reagent or by direct activation,where the solvent transporting the activating reagent also serves todissolve the protective layer. In the second case, the dissolvedprotective layer is then removed by subsequent rinses. Finally, abioreagent or biological reagent, such as a polymer, protein, peptide,or enzyme, is bound to the surface of the target areas. The binding ofthe bioreagent is facilitated by the activated reagent already bound tothe target areas. In the case where there is activated reagent coated bythe protective layer, the bioreagent may be added to the surface byeither removing the protective layer and then adding bioreagent to thetarget area or by direct binding, where the solvent transporting thebioreagent also serves to dissolve the protective layer.

An advantage to the above target or target array manufacturing processis that the targets, once coated with the protective layer, may bestored for extended periods of time and then used at the discretion ofthe consumer. Another advantage of the target array is to provide aconfined reaction surface for analyte processing and manipulation ofimpurities.

In yet another similar embodiment, mass spectrometer target surface orsurfaces are derivatized with chemicals found to enhance samplepreparation through promoting the formation of crystals of matrices usedin the practice of MALDI. Such matrix crystal “seeding” is foundinvaluable in the automation of the entire sample preparation process,enabling the production of highly reproducible samples over the entirearea of the target.

High Throughput Machine

The individual components described herein come together to form asingle, integrated system capable of high-throughput analysis ofanalytes retrieved from biological media. Fundamental analyses beginwith verifying the primary structure, i.e., sequence of analytes.Oftentimes, a single high-accuracy determination of molecular weight issufficient to verify the primary structure of analytes. If higherprecision is required in verifying the primary structure of an analyte,it is convenient to mass map the analyte (after retrieval) usingchemically/enzymatically active mass spectrometer targets. During suchmapping procedures, an analyte is digested using high specificitycleavage reagents to produce a multitude of signals when analyzed usingmass spectrometry. When viewed as a group, these signals are able toverify primary structure with greater precision and redundancy than asingle mass determination. Alternatively, these data can be used tosearch databases for variants of an analyte that differ largely fromthat predicted for a normal analyte, e.g., splice variants.

In a similar embodiment, other variants of analytes are mapped toelucidate the nature, location and origin of the variation. Analytes andvariants present in a single sample are co-extracted from biologicalmedia using a common affinity reagent localized in the microcolumn andare simultaneously subjected to mapping on an activated massspectrometer target or target array. Because most analyte variants willshare a large degree of homology with the normal analyte, most mappingsignals will be common between the analyte and variant. However,uncommon, or mass-shifted, signals will also be present within themapping data. Using these differential data, in combination withknowledge of the cleavage agent and information of the primary,tertiary, quanternary structures of the normal analyte, it is possibleto elucidate the site of the variation. Furthermore, using knowledge ofmass differences between component residues of the analyte (e.g., massdifference between amino acids in proteins or nucleic acids in DNA/RNA)and accurate determination of the mass-shifts, it is possible todetermine the transposition that created the variant. Such analyses areof great value in elucidating, e.g., point mutations present in proteinsor polymorphisms present in nucleic acids. Likewise, knowledge ofmolecular weights of potential modifying groups (e.g., glycans,phosphates, methyl, formyl and the like) can be used in combination withmapping data to elucidate the sites and nature of chemical modificationsof the analyte. Finally, reactive targets designed to address specificmodifications can be used in the integrated system for the determinationof the quantity (number) of modifying moieties by cleaving them from theanalyte and viewing subsequent mass shifts in mass spectra.

In another embodiment, the present invention is used in thehigh-throughput quantitation of specific analytes present in biologicalmedia. Using this process, analytes and internal references(analyte-like species) are simultaneously retrieved from biologicalmedia and processed through to mass spectrometry. In the same parallelprocessing operation, standard samples are analyzed to produce workingcurves equating analyte signal with the amount of analyte present in thebiological media. The amount of analyte present in each sample can thenbe either judged as elevated relative to other samples, or determinedabsolutely using the working curve.

In a further embodiment, the present invention is used in determiningthe interacting partners involved in protein-ligand interactions.Essentially, affinity microcolumns are derivatized with ligands ofinterest with the intention of screening biological media forinteracting partners. The ligands act as affinity receptors capable ofselectively isolating analytes from the biological media. Once isolated,the analytes are subjected to mass spectrometry for identification.Oftentimes, direct mass spectrometric analysis, and a knowledge ofcomponents present in the biological media, is sufficient to identifyretained analytes via direct molecular weight determination.Alternatively, unknown analytes are subjected to digestion usingchemically/enzymatically active targets and the resulting fragments(e.g., proteolytic fragments) subjected to mass spectrometry. Theaccurately determined molecular weights of the fragments (and knowledgeof cleavage specificity) are then used to fuel genomic or proteindatabase searches capable of identifying the analytes.

In a similar embodiment, protein-ligand interactions are investigated bydesigning an affinity reagent to target a specific protein that initself retains other analytes. In this manner, protein complexes areretrieved from biological media by targeting one of their constituents.Using the aforementioned analytical approaches, the identity and natureof the components of the complex are then delineated.

Specific embodiments in accordance with the present inventions will nowbe described in detail. These examples are intended to be illustrative,and the invention is not limited to the materials, methods or apparatusset forth in these embodiments.

EXAMPLES Affinity Microcolumn Manufacture

Below are described the directed formation of the preferred embodimentsof biologically sensitive affinity-ligated microcolumns capable of highthrough-put via efficient affinity capture, release and rapid, sensitiveand accurate mass spectrometric analysis of specific or non-specificallytargeted analytes. The below examples describe numerous approaches,architectures and device deliveries to provide stable configurations inbiological rich environments.

Example 1 Porous Glass Molecular Trap Preparation Using MetalMold-Graphite Spray Release

Porous glass molecular traps are metered to the specifications ofcommercially available wide-bore P-200 pipettor tips using annealingmolds made of stainless steel (100-1000 holes of 0.071 inch (entrance)per mold; 2-degree taper, polished and treated with graphite releaseagent). The molds are loaded with soda lime glass spherical beads(150-200 μm; 75% SiO₂, 15% Na₂O and 10% CaO), and annealing is achievedin an argon-backfilled furnace by ramping the temperature from 772° C.(equilibrated, t=0) to 800° C. (t=3 minutes; one-minute equilibration).Upon completion of the ramp-anneal, the molds are immediately removedfrom the oven and the porous glass molecular traps extracted from themolds. This process typically yields porous glass molecular traps withhigh-flow characteristics and appropriate bore and taper to fit theentrance of the wide-bore P-200 pipette tips (room temperature porousglass molecular traps dimensions: 0.061 in. (entrance), 0.092 in.(length), 2-degree taper).

In a preferred embodiment, the molecular traps are then inserted intothe top of the pipette tip and allowed to locate to the bottom section(narrow section). The molecular traps are seated by the application of asufficient amount of pressure applied from the top. The pipette tip maybe heated prior insertion of the molecular trap to aid in the seatingprocess.

Sample mass spectra using an affinity microcolumn prepared according tothis example are illustrated in FIG. 2-5.

Example 2 Porous Glass Molecular Trap Manufacture Using CeramicMolds—Pyrex

Ceramic molds are described to manufacture porous Pyrex glass moleculartraps. Porous glass molecular traps are metered to the specifications ofcommercially available wide-bore P-200 pipettor tips using ceramicannealing molds. Zircar type-R plates (four by six by one-quarterinches) were purchase from Zircar Corporation (Florida, N.Y.) and wereend-mill surfaced for maximum flatness and CNC machined (2100 holes,0.0625 inch bottom cut, 2-degree taper). Four of the ceramic moldsloaded with ball-milled powdered borosilicate “Pyrex” (size ranging from4 μm to 300 μm; 81% SiO₂, 4% Na₂O, 0.5% K₂O, 13% B₂O₃ and 2% Al₂O₃),were stacked in a furnace where they underwent initial temperatureequilibration, using a slow temperature up-ramp (60 minutes) to belowthe Pyrex softening point (816° C.). The molds were then equilibratedfor thirty minutes prior to being ramped to about the Pyrex softeningpoint (821° C.), which was then maintained for about thirty minutes toform porous glass molecular traps. For concerted heat treatment (usingenhanced silica glass); the molds were down-ramped to 708° C. where theywere maintained for 2 to 20 hours (depending on the amount of etchingdesired) prior to a slow final temperature ramp down for removal. Whenimmediate use is required, the molds are slowly temperature down-rampedto a reasonable retrieval temperature (generally 300° C.) to avoid glasscracking. This process typically yields porous Pyrex glass moleculartraps with high-flow characteristics and appropriate bore and taper tofit the entrance of the wide-bore P-200 pipette tips (room temperatureporous glass molecular traps dimensions: 0.0625 in. (entrance), 0.130in. (length), 2-degree taper).

Example 3 Porous Silica Glass Molecular Trap Manufacture Using CeramicMolds—Vycor

The ceramic molds described in EXAMPLE 2 are used to manufacture porousVycor glass molecular traps. Zircar type-R ceramic molds were loadedwith powdered “Vycor” (Corning Corporation), porous Vycor (CorningCorporation), controlled porous glass (Controlled Porous Glass, Inc.,NJ), (size ranging from 4 μm to 300 μm, depending upon desiredflow-through characteristics; 96% SiO₂), and were stacked in a furnacewhere they underwent initial temperature equilibration, using a slowtemperature up-ramp (90 minutes) to below the Vycor softening point(1500° C.), they are then equilibrated for thirty minutes prior to beingramped up to about the Vycor softening point (1530° C.), which wasmaintained to form porous glass molecular traps. The molds were slowlytemperature down-ramped to a reasonable retrieval temperature (generallyabout 300° C.) to avoid glass cracking. This process typically yieldsporous Vycor glass molecular traps with high-flow characteristics andappropriate bore and taper to fit the entrance of the wide-bore P-200pipette tips.

Example 4 Porous Silica Molecular Trap Manufacture Using CeramicMolds—Silica Gel or Fused Silica

The ceramic molds described in EXAMPLE 2 are used to manufacture porousglass molecular traps using silica gel or fused silica. Zircar type-Rceramic molds were loaded with powdered porous silica gel (SigmaChemical Company) or fused silica (Corning Corporation) (sizes rangingfrom 4 μm to 300 μm depending upon desired flow-through characteristics;100% SiO₂), and were stacked in a furnace where they underwent initialtemperature equilibration, using a slow temperature up-ramp (90 minutes)to below the silica softening point (1550° C.) where they areequilibrated for thirty minutes prior to being ramped to about thesilica softening point (1585° C.), which is then maintained to formporous silica molecular traps. The molds were slowly temperaturedown-ramped to a reasonable retrieval temperature (generally about 300°C.) to avoid silica cracking, This process typically yields poroussilica molecular traps with high-flow characteristics and appropriatebore and taper to fit the entrance of the wide-bore P-200 pipette tips.

Example 5 Etched Porous Silica Molecular Traps

Hyper-porous porous silica molecular traps formation is accomplished byexposing porous silica molecular traps formed in Example 4 to variouselectrochemical etching conditions. For example, porous silica moleculartraps are placed in a solution of aqueous HF (25-50%) in absoluteethanol while applying an etching current (120-200 mA/cm²) andirradiation (150 mW/cm²) for one to twenty minutes thereby creatingporous silicon hydride surfaces.

Example 6 Porous Powdered Metal Molecular Trap Manufacture Using MetalMold

Porous metal molecular traps are metered to the specifications ofcommercially available wide-bore P-200 pipettor tips. A metal mold ismachined, having either a reverse incline (relative to the previousmolds, i.e., narrow at the top) or the same directional taper, andcontaining a slight raised lip (for required overfill) around theopenings located at the bottom of the mold (for porous powdered metalexit by a pushpin). The mold rests upon a removable bottom plate, isloaded with powdered metal (e.g., brass, copper, silver, gold), andpressure (commensurate to form individual welds between metal particles)is applied to the throated/raised powdered metal surface via an Arborpress equipped with a complementary push-pin for each hole. After thepowdered metal molecular traps are formed, the bottom plate is removed,and a pushpin is applied to the narrow end of the hole to release theporous powdered metal molecular trap from the mold. Porous powderedmetal molecular traps are oven annealed to enhance architecturalstability and form metal oxides for organo-functionalization.

It should be recognized that the molecular traps may be made from avariety of different materials and or different combinations ofdifferent materials and still fall within the scope of the presentinvention. The only limiting requirement is that the materials must beable to bind affinity reagents, or chemically modified to bind affinityreagents, to their surfaces. The most preferred materials orcombinations of materials have high surface areas or are capable ofbeing modified to have high surface areas.

While the above examples utilize a tapered profile for the microlumns,non-tapered or shapes, such as cylindrical columns, may be used andstill fall within the scope of the present invention. In deed, for highthroughput embodiments, the non-tapered form is preferred since thetaper slows the manufacturing process.

Chemical Activation of the Molecular Traps Example 7 SilaneCoating/Functionalization of Porous Glass Molecular Traps

Porous molecular traps formed via EXAMPLES 1-6, or other equivalentprocesses, may undergo pretreatment conditioning combining variouswater, mineral acid treatments, and concomitant water rinses and dryingprior to chemical derivatization (silanization). For example, porousglass molecular traps are leached by suspending them in distilled orpurified water (generally ten fold by volume) under elevatedtemperatures (typically below the boiling point of water) with orbitalshaking or 1 to 24 hours, with (or without) two to three change outs offresh water. All water is then removed and two subsequent acidtreatments applied. First, 1N hydrochloric acid in water is applied (tenfold) over the porous glass molecular traps, at elevated temperatures atime, such as one to two hours, and water rinsed until a neutral (ornear neutral) pH is obtained. Second, 1N nitric acid in water is carriedout as performed with the first acid treatment. The porous glassmolecular traps are dried either at room temperature or in a vacuum oven(100° C., 1 atm, overnight).

The dried/pretreated porous glass molecular traps are then chemicallyactivated, such as by coating them with a silanating reagent, e.g. 10%silanating agent, ca. N-[3-(trimethoxysily)propyl]ethylenediamine, inanhydrous toluene under reflux with orbital shaking overnight, toproduce functionalized porous molecular traps. The silane coated porousmolecular traps are allowed to cool to room temperature, rinsed inmethanol under reflux, followed by room temperature rinses, until thesupernatant is negative to ninhydrin and/or trinitrobenzoic acid (tnbs)analysis. Other, useful silanating reagents include, but are not limitedto, 3-(trimethoxysily)propyl acrylate, 3-(trimethoxysily)propyl anmine,N-[3-(trimethoxysily)propyl]aniline,N1[3-(trimethoxysily)propyl]diethylenetriamine,N-[3-(trimethoxysily)propyl]ethylenediamine, 3-(trimethoxysily)propylmethacrylate, [3-(trimethoxysily)propyl]octadecyldimethlammoniumchloride, N-[3-(trimethoxysily)propyl]polyethylenimine,N1-[3-(trimethoxysily)propyl]carboxylic acid,aminopropyltriethoxysilane, 3-glycidoxypropyltrimethoxysilane,thiolpropyltrimethoxysilane, chloropropyltrimethoxysilane,octadecyltrimethoxysilane, octadecyltrichiorosilan,O[2-(trimethoxysily)ethyl]O′-methylpolyethylene glycol, silyl aldehyde,and the like.

These reactions may be accentuated by base catalysis, such as usingtriethylamine (TEA) added to the reactions or by performing them underpressure by applied vacuum (ca. 1 atm).

Microcolumn Housing (Pipette) Manufacture

In the preferred embodiment, where non-tapered molecular traps are used,micropipettes, which are used as the microcolumn housing are modified asfollows:

Microcolumn housings, such as micropipette tips are formed, such as byinjection molding or other means well known in the arts. The distal endsof the housings are modified from ordinary micropipettes by providing atleast one inset projection, such as a ledge. In one embodiment, theprojection is a continuous ledge that following the inner diameter ofthe distal end of the housing. However, in a more preferred embodimentthe at least one inset projection is a single tang or triangularprojection, more preferably, three tangs, and even more preferably sixtangs. Thus, when the molecular traps are loaded into the housing, eachrests upon the at least one inset projection. After the molecular trapsare loaded into the housings, a portion of the sides of the housingdirectly adjacent to the molecular traps are crimped inward to a degreewhere there is slight frictional contact with the molecular traps. Caremust be taken not to over crimp since that would destroy the containedmolecular traps. In a preferred embodiment, the crimping is accomplishedby heating the outer surface of the housing while cooling the containedmolecular trap with a flow of cool gas (an inert gas is preferred). Theheated housing is then forced inward using a gradual taper in thebeating mold, thereby crimping the housing to the molecular traps.

Batch Functionality Example 8 Batch Direct Activation/Derivatization ofFunctionalized Porous Molecular Traps

Direct batch activation/derivatization of microcolumns targets anyreactive chemical group that will couple to the representative affinityligand and provide an environmentally stable linkage. Batchactivation/derivation approaches proceed in a myriad of ways, targetingactivation/conjugation chemistries for biological modification witheither organic and inorganic reagents, for example: primary amines,carboxylic acids, thiol groups, sulfhydryls hydroxyls, allyl groups,azides, aldehydes, hydrazides, maleimides; triazine and the like, thatcan be activated with homobifunctional, heterofunctional or polymericreagents.

The instant example illustrates one of these approaches by incorporatingglutaraldehyde activation followed by ligand coupling through primaryamine groups with aldehyde groups on the glutaraldehyde-porous moleculartrap. First, amine functionalized molecular traps are coupled toglutaraldehye (a 25% solution in 0.10 M sodium phosphate, pH 7.8, 100 mMNaCl buffer) using sodium cyanobohydride (10 mg/mL) mediated coupling(reaction times ranging from an hour to overnight). After several rinseswith the phosphate buffer, the activated aldehyde groups on theglutaraldehyde matrix are incubated with the ligand of interest, such asa protein antibody. Uncoupled (excess) ligand is removed by extensiverinsing with HBS buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005%Surfactant P20). This derivatization process yields molecular traps witha high analyte binding capacity. The molecular traps are then packedinto the P-200 pipettor tips.

Example 9 Batch Amine Activation, Amplification, Reactivation andDerivatization of Functionalized Porous Molecular Traps

amine->CHO->poly-amine->CHO->Ab

Microcolumn activation with amplification allows another approach foraddressable ligand coupling. To create an amplified amine surface, midto high-molecular weight amine-polymers, such as polylysine,aminodextran or amine modified starburst dendrimers, are cross-linked tothe functionalized/activated porous molecular traps surface through avariety of appropriate chemistries. A glutaraldehyde activated aminemicrocolumn is incubated with polylysine in a sodium cyanoborohydratemediated coupling. This polyamine microcolumn is then re-activated usingglutaraldehyde, water rinsed and protein ligand conjugated.

Example 10 Damping of Nonspecific Binding Via Batch Surface AmineModification Activation/Derivatization of Functionalized PorousMolecular Traps

amine->CHO->poly-amine->NHS/EDC->polyCOOH->NHS/EDC->LIGAND

Another approach to ligand immobilization is through coupling ofligand-borne carboxyl groups to surface amines. An opportune way ofcreating an amplified amine surface is through cross-linkinghigh-molecular weight polylysine, aminodextran, amine modified starburstdendrimer or polyacrylhydrazide to the functionalized porous moleculartraps surface followed by distal carboxyl generation through a varietyof chemistries, such as EDC-mediated linkage of carboxyl-terminatedcross-linkers (e.g. succinic or glutaric anhydride) or bisoxiraneactivation (resulting in an epoxide-activated surface). These carboxylmicrocolumns are then used to immobilize low-pI protein ligands via ca.,EDC/NHS-mediated activation/coupling. Specifically, polylysinemicrocolumns are incubated in bulk in succinic (or glutaric) anhydride(100 mg/mL anhydride in 0.2M sodium acetate pH 4-5) with the reactionallowed to proceed to completion (i.e., negative tnbs and ninhydrinamine testing) generally for two to four hours, while pH is maintainedby dilute hydrochloric acid addition. The carboxy microcolumns are thenactivated with 100 mM NHS and 100 mM EDC in water for 10-20 minutes atroom temperature, water rinsed and a slight vacuum is applied prior toprotein ligand coupling (antibody 1-10 mg/mL HBS buffer pH 7.5,overnight 4° C., shaker).

Example 11 Batch Polymer Modified Carboxylic AcidActivation/Derivatization of Functionalized Porous Molecular Traps

Another approach involves surface amplification with water-solublecarboxyl containing polymers (carboxymethyl dextrans, carboxymethylagaroses, carboxymethyl celluloses, carboxymethylamylose, polyglutamicacid, poly acrylic acids, carboxyl modified starburst dendrimers) likecarboxymethyl dextran, followed by coupling of ligands, such as viainteraction of primary amine groups with the carboxyl groups on thedextran matrix. First, the amine functionalized microcolumns are coupledto 15 kDa carboxymethyl dextran (CMD) executed in 100 mM sodiumphosphate, pH 4.8, 100 mM NaCl buffer, via EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) mediated coupling.After several rinses with the phosphate buffer, the carboxyl groups onthe dextran matrix are activated with a mixture of EDC/N-hydroxysuccinimide (NHS; 100 mM each, in H₂O) and incubated with the ligand ofinterest, such as a protein antibody. Uncoupled (excess) antibody isremoved by extensive rinsing with HBS buffer (10 mM HEPES pH 7.4, 0.15 MNaCl, 0.005% Surfactant P20), after which the molecular traps are packedinto the P-200 pipettor tips. This derivatization process yieldsmicrocolumns with a higher binding capacity, compared to microcolumnsfor which no amplification layer (the CMD matrix) is used. Although theinitial amino-silation activation step results in amine surfaces (defacto a “functional surface”), it is preferred to amplify the surface inorder to increase distance off the binding surface, loading capacity,and ensure a uniform (homogeneous) surface coating throughout the entiremicrocolumn (thereby reducing potential interactions that may occur withthe glass substrate and result in non-specific binding).

Example 12 Batch Carboxymethyl Dextran Carbonyldiimidizaole (CDI)Activation/Derivatization Of Functionalized Porous Molecular Traps

In yet another approach to ligand immobilization, the EDC/NHS activationprocedure of the CMD layer is replaced by an activation withN,N′-carbonyl diimidazole (CDI). This activation results in carboxyl andhydroxyl groups present in the CMD being converted to an imidazoylcarbamate intermediate capable of reacting with primary amines orsulfhydryls present in the affinity ligands. For instance, CMDmicrocolumns are rinsed with acetone followed by dimethylsulfoxide(DMSO). A 100 mg/mL solution of CDI in DMSO is then overlain above theCMD microcolumns, a vacuum applied, and the activation allowed toproceed from two hours to overnight on an orbital shaker. The CDIactivated affinity pipettes are extensively rinsed in DMSO followed byacetone to form a CDT-activated CMD microcolumn. Importantly, theCDI-activated media is known to be reasonably stable, with shelf liveson the order of months given proper storage conditions (i.e., (wet)isopropanol or (dry) inert gas or vacuum). Thus, the CDI can act as astable activator of the microcolumns and increase the longevity forpre-activated supply. Alternatively, the CDT-activated matrix isincubated with the protein antibody of interest. Uncoupled (excess)antibody is removed by extensive rinsing with HBS buffer (10 mM HEPES pH7.4, 0.15 M NaCl, 0.005% Surfactant P20), after which the moleculartraps are packed into the P-200 pipettor tips.

Example 13 Batch In-Situ Polymerization or Co-Polymerization Via SurfacePolymerization of Organo Functionalized Porous Glass Molecular Trap

In this example, organo-functionalized porous glass molecular traps(c.a., methacryltrimethoxysilanated) are in-situ polymerized via freeradical initiation. Typical co-polymerization monomers used include butare not limited to allyl dextran, allylamine allyl glycidyl ether,acrylic acid, and vinyldimethyl azlactone which can incorporatecross-linking agents, such as methylene-bis(acrylamine), using typicalinitiating reagents such as tetramethylenediamine (TEMED) orN,N,N′,N′-tetramethyl-1,2-diaminoethane and ammonium persulfate inwater. The degree of cross-linking and polymer size is controlled byamount of initiator, polymerization time, and available monomers fortermination. This surface reaction results in functional groupsabounding within the polymeric matrix, which covers the molecular trapsurface. These groups are then available for activation/conjugation toligands or subjected to additional activation and amplification prior toactivation and ligand coupling.

Example 14 Metal Chelator Modified Porous Glass Molecular Traps

The incorporation of metal chelators into porous glass molecular trap isuseful for metal binding biomolecule retrieval. Amine or polyaminemicrocolumns, prepared as in previous examples, are overlain in 0.1Mphosphate at pH 7 and evacuated using a rotary evaporater. Primary aminesurfaces are then directly coupled to bifunctional chelating agents suchas ethylenediaminetetraacetic dianhydride (EDTA-DA), anddiethylenetriaminepentaacetic dianhydride (DTPA-DA) at 20-100 mg/mL toform metal-chelating arms (TED and EDTA respectibly) able to bindtightly metals in a coordination complex for biomolecule trapping.

Alternatively, metal-chelators such as ethylenediaminetetaphosphoricacid (EDTPA), 1,4,7,10-tetracyclononane-N,N′,N″-triacetic acid (NOTA),1,4,7,10-tetraacacyclododecane-N,N′, N″,N′″-tetracetic acid (DOTA),1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA),1,2-bis(2-aminophenoxy)ethane-N,N,N′N′ tetraacetic acid (BAPTA),N,N(biscarboxymethyl) L-lysine are solution activated using for instanceNHS/EDC (100 mM each, 0.1 M phosphate, pH 7) mediation to couplecarboxyl groups to localized surface primary amines.

Example 15 Formation of Biomimic Ligands, Preparation of BatchDye-Functionalized Porous Glass Molecular Traps

Synthetic dyes are useful affinity ligands by virtue of their reactivitywith a wide variety of biological materials. Reactive dyes encompassingbut not limited to triazine dyes (i.e., reactive black, reactive blues,reactive browns, reactive greens, reactive oranges, reactive reds,reactive yellows, etc.), for instance, have been linked toamino-microcolumns using a simple approach. The triazine dye ofinterest, ca. Cibacron Blue at 50 mg/mL dissolved in distilled water, isadded to amino microcolumns in a round bottom flask and allowed to reactovernight under low reflux. After ligand coupling, the biomimicmicrocolumns are rinsed with water and incubated with 1N salt solutionunder low reflux for thirty minutes. After extensive water rinses, thebiomimic microcolumns are dried either in air or under vacuum at 90° C.

Example 16 Formation of Ion Exchange-Functionalized Porous GlassMolecular Traps in Batch

The incorporation of ion exchange media into porous glass molecular trapis useful for bio-molecule binding, retrieval and analysis or in samplecleanup as in desalting surfactant (SDS removal). In this instance, theamine or polyamine microcolumns, are activated targeting for examplecarbohydrate hydroxyl groups contained within dextran ion exchange media(e.g., dextan-sulfate, diethylaminoethyl-dextran (DEAE-dextran),heparin-sulfate, carboxymethyl dextran (generation described previouslyin EXAMPLE 10). Primary amine surfaces are activated with bifunctional(or activating) agents such as triazine, diglycidoxyether (oxiranines orepoxides) prior to dextran ion exchange carrier coupling. Likewiseepoxide-functionalized microcolumns generated from initialsilylanization reactions or via co-polymerization are used forderivatization.

Alternatively, amine microcolumns are incubated/coupled to activateddextran carriers. A further example is the use of aldehyde groups ondextran carriers generated by sodium meta periodate oxidation of dextran(vicinal diols) ion exchange carriers. Specifically, dextran ionexchange media (10-100 mg/mL) is incubated in the dark in 1M sodium metaperiodate (in water), for two hours after which, the oxidized dextranion exchange media is either precipitated in organic (ethanol/ether) ordialyzed in water overnight. Oxidized dextran ion exchange media is thenincubated with amine or polyamine microcolumns (0.1 M phosphate, pH7.5).

Example 17 Protein Immobilization to Microcolumns

Proteins with specific binding properties have specialized uses asreceptors and ligands in immuno-chemistry. These affinity proteinsinclude but are not limited to protein A, protein G, avidin andstreptavidin reagents. Protein ligands, specifically protein A or G, areincorporated into porous glass molecular traps, for distal constantregion (Fc) antibody capture or capture/conjugation with subsequentcross-linking prior to use as biomolecule retrieval agents.Avidin/biotin systems are used as alternative tethering agents whichwhen coupled to antibody or analyte create conjugated agents with notonly strong binding properties but with an amplified motif.

For example, amine or polyamine microcolumns, prepared as in previousexamples, are overlain in 0.1M phosphate at pH 7 and evacuated using arotary evaporator. Primary amine surfaces are then glutaraldhydeactivated (25%, 4 to 24 hours), rinsed, and directly coupled tofunctional protein agents (or amine or hydrazide modified biotin) (1-10mg/mL in HBS-EP) in a sodium cyanoborohydride (0.1 mg/mL) mediatedcoupling.

Example 18 Protein Modification/Digestion Using Microcolumns

Biomolecules with specific modifying or digestion properties are used asreceptors and ligands in biochemistry. These biomolecules include, butare not limited to, proteins, peptides, enzymes, catalytic antibodies,and the like.

For example, trypsin modified microcolumn is prepared as in previousexamples. When activated, the trypsin will make specific digests ateither arginine or lysine points in amino acid sequences of proteins,such as a targeted analyte protein.

Alternately, other receptors may be used to synergized two biomoleculesinto a single entity, such as phosphorilization of an analyte byimmobilized kinase.

Target Manufacture

The current target designs used by the commercial manufactures do notadequately support the rigorous and consistent use of arrayed and/orbioreactive targets. It is of great importance for these targets toachieve a high degree of inter-sample reproducibility.

There are at least three targets designs capable of correcting theproblems encountered using the current design targets. These designs areas follows:

Targets Utilizing Plateaus

In a first embodiment of targets according to the present invention,there are “matched set” targets wherein a volume of analyte can beapplied to a strictly defined area without the worry of spreading beyondthe active area of the target, Volumes of analyte will meniscus on thetop of a plateau formed by the target and, if correctly designed, willnot seep down the sides of the plateau. Because of this meniscus action,the analyte will generally be in an aliquot volume of ˜2-3 μL. Thisvolume will fully cover the ˜4 mm² area of the target completely, withlittle concern of overrunning the enzymatically-active area. Erraticdigestion due to exposing the analyte to varying amounts of enzyme istherefore eliminated by fully covering (only) the active surface of thetarget. In addition, the ˜2-3 μL volume will not evaporate if thetargets are placed in an appropriately humidified incubator. Erraticdigestion because of sample drying is therefore eliminated bymaintaining the constant sample volume on the plateau.

In manufacture, there are plateaus milled into metal-base targets thatfit into a larger (stainless steel) target that is ultimately introducedinto the instrument. The height of the plateaus is determinedempirically, with the criteria of finding the minimum height necessaryto avoid analyte spreading. A height of ˜0.5 millimeter is usuallyadequate; a height that can be easily introduced into the accelerationregion of all MALDI-TOF instruments (minimum clearance ˜2 mm). Theoverall dimensions of the plateaus are such to adequately confineanalyte without introducing instrumental artifacts into the analysis.

A second embodiment uses thin-gauge metal targets that fit into theinstruments via a tongue-in-groove design. Because of the thin gauge ofthe targets, and the necessity of having a grooved mounting bracket, itis impractical to mill (machine) these targets. Therefore, a pressmethod is used (pressing the targets from the backside) to create theplateaus. Note, the pressing method requires the machining of anappropriate die.

The appropriate base material is determined empirically forcompatibility with solvents used during manufacture, gold-coatingproperties and vacuum compatibility. Initially, base targets are made ofpolystyrene, which is compatible with manufacturing solvents, can besputter-coated with high uniformity and has low out-gassing properties.However, other polymeric materials may be used. An advantage of thepolymer-based targets is that they can be manufactured in large quantityat little expense.

The criteria for success with this type of target is to determine anoptimum plateau geometry that: 1) adequately confines sample volumes toan exact surface area, 2) does not detract from the performance of theinstrument, and 3) is easy and cost-effective to mass-produce.

Hydrophobic/Hydrophilic Contrast of Targets or Target Arrays

A second embodiment design for high-reproducibility bioreactive targetsis to contrast enzymatically active sites with a hydrophobic mediacapable of confining the analytical volume to only the active area. Inthis approach, the enzyme is immobilized to gold/DSP/enzyme spotslocated on top of a generally hydrophobic target. The simplest way tomanufacture the contrast devices is to construct them out of ahydrophobic material such as Teflon. Gold sample areas are placed on thetarget by a masking/sputter-coat process. The gold areas are thenderivatized using our normal activation/immobilization methods. Oneconcern regarding the Teflon device is that of surface charging in themass spectrometer. Such effects (charging of the sample stage by removalof ions) are always a concern in mass spectrometry design. MALDI-TOFanalyses have been done from sample targets made of non-conductingmaterials (e.g., quartz targets or gels) with only minor effects due tosample charging. These effects are even further minimized using thedelayed-extraction methods available on all commercial MALDI-TOFinstrumentation. However, any perturbations to mass spectrometerperformance due to the targets may be eliminated by such methods asincluding electrical linking of the enzymatically-active surfaces in themasking/gold-coating process.

Another possibility for contrast devices is to derivatize metal-basetargets to create a hydrophobic surface onto which hydrophilicenzymatically-active areas are constructed. Manufacturing protocols forthis approach are more involved than the previous approach. It isnecessary to follow a multi-step protocol to first coat the target withthe hydrophobic media and then gold-coat a pattern of sample spots ontothe hydrophobic coat using a masking process. The process involves: 1)Sputter coating of entire targets with gold, 2) Activation with DSP (ifnecessary), 3) Immobilization of hydrophobic compound, 4) Re-sputtertarget using pattern mask, and 5) Activate fresh gold and immobilizeenzymes. An example using this process is to gold-coat a target andderivatize with 1-octadecanethiol (skip DSP activation). This processhas been used to create C-18 derivatized surfaces. These surfaces arehighly-hydrophobic as can be readily observed by beading of water. Oncethe target is derivatized with hydrophobic compound, a pattern of freshgold is applied by masking and sputter-coating. The fresh gold is thenactivated and derivatized using e.g., the DSP/dextran/enzymeimmobilization procedure. The process results in hydrophilic,enzymatically-activated spots surrounded by a hydrophobic media. Toenhance visual contrast between active and hydrophobic areas,hydrophobic dyes containing a single primary amine and no otherchemically-reactive group (e.g., Fast Violet B, Methylene Violet 3RAX)can be used in place of the C-18 layer. Likewise, other classes of dyesor chromaphoric hydrophobic compounds can be used to create more visualcontrast between activated and hydrophobic areas of the targets. Inpreferred embodiment, hydrophillic mecaptoundecanoic acid dissolved inisopropanol is flash micro-deposited onto the arrayed targets, dried,and covered with octadecyl mercaptan rapidly forming contrasted targetarrays. Contrasting can be reversed for hydrophobic analytes, or anycombination thereof, including mixed SAM applications.

The criteria for success with this type of target is to find generalderivatization methods (for high contrast targets) that are not toolabor intensive or costly to prohibit large-scale manufacture.

Individual Targets (Inserts)

A third target embodiment design is that of using individual areas(inserts) that after digestion can be inserted into a base that fitsinto the commercial mass spectrometers. This approach emulates the“matched set” targets described above. The inserts will be of eitherplateau or contrast design, and will be economical to derivatize usinghigher cost enzymes (by using smaller volumes of higher concentrationreagents). These targets (even when derivatized with low-cost enzymes)are very economical in terms of cost in research applications where theycan be used one at a time.

The necessary limitation for success with this type of target is toconstruct “matched set” design targets as inserts fitting into thetargets accepted by commercial instrumentation.

Biochips, and Small Array Targets

Another embodiment of the target arrays is incorporating bioreactivesurfaces into/onto existing chip-based bioanalytical platforms.Micro-channel devices (chips) are now used in protein analysis and areideal platforms for biomolecular separation (in one dimension) followedby enzymatic processing (by driving separated biomolecule in a seconddimension over an enzymatically-active area) and MALDI-TOF analysisdirectly from the chip.

Other Active Surfaces, Derivatization Methods and Assays AmplifyingMedia

In an another alternate embodiment surface amplifying media are used toincrease the activity of the target array or the enzymatically-activetargets. An example amplification is performed using solution basedpolymeric amplification techniques, either single step activatedcouplings, or in situ induced polymerization events. These modifiedsurfaces display definite charge differences using either carboxylicacid or amine amplifying reagents (which are in themselves eitheractivated or activatable).

General Enzyme Immobilization Kits

A significant product according to the present invention is afully-activated General Enzyme Immobilization Kit The kits are comprisedof a number of affinity micro-pipettes containing affinity microcolumns,activated or activatable targets or target arrays (whether amplified ornot), and pre-made buffers. The purpose of the kits is to have theend-user derivatize targets in-house using proprietary enzymes, therebyeliminating shipment of reagents to the manufacturer for immobilization.

Ion-Exchange Surfaces

In still yet another embodiment, the targets or target arrays may bederivatized with carboxymethyl dextran (CMD) capable of a cationexchange process, which ultimately leads to higher quality MALDI-TOFspectra during the analysis of proteins in the presence of sodiated andpotassiated buffers. Using the CMD targets, unwanted cations arescavenged from solution and replaced with protons or ammonium ions,dependent on which cation is used to pre-charge the targets.

Cation Exchange (CE) Targets

The value of CE surfaces has already been described; reduction ofunwanted cations in buffer solutions to reduced signal heterogeneitythereby increasing mass spectral sensitivity (homogenization of signal).Reduction of alkali cations, using the CE surfaces, is also capable ofimproving matrix homogeneity during sample preparation. These attributesare of benefit to the MALDI-TOF analysis of both protein and nucleicacids. In one embodiment of a CE surface uses amplified surfaces of 500kDa CMD. The 500 kDa CMD is chosen because of a higher exchange capacitythan lower molecular weight CMD. It is estimated that approximately 10picomole of CMD can be immobilized to the surface of a 4 mm² target.Considering that each strand of CMD will have ˜1,000 valence sites forexchange (˜30% of monomeric dextran converted to CMD), an exchangecapacity of 10 nanomole is estimated for the targets, equating to a 1 μLaliquot of 10 mM alkali salt. However, the exchange properties of theCMD targets will have to be evaluated to determine the overalleffectiveness in sample clean up. They are effective in alkali metalremoval using a short single stranded DNA molecular (e.g., d(T)₈) mixedin the presence varying concentrations (0.1-10 mM) of buffer salts. DNAis chosen for the test assay because of the high propensity to retainalkali metals as counterions to the negatively charge phosphatebackbone.

Using standard CMD targets it is likely to reach an exchange limit belowthe buffer concentration ranges used in a number of biologicalapplications. Stronger CE functionalities (e.g., sulfonate groupscreated by treatment of dextran with chlorosulfonic acid) and methodscapable of greater surface amplification (application of polymericresins to create macroscopic (10-100 micrometer surfaces) may be used inorder to raise the exchange capacity of the targets to a level capableof reducing deleterious effects experienced during the analysis ofbiomolecules present in moderate-high strength buffers.

Anion Exchange (AF) Targets or Target Arrays

Anion exchange targets or target arrays are a still further embodimentuseful in sample cleanup by scavenging various anionic detergents fromsolution. Specifically, the presence of sodium dodecylsulfate (SDS) inprotein solutions has always been of concern during MALDI-TOF analyses.SDS is generally disruptive of the crystal formation required for mostof the MALDI matrices to function, and therefore needs to be removedfrom solution as part of sample preparation. Another anion that possiblyhas a deleterious effect on the MALDI process is phosphate ion.

Polylysine-amplified surfaces have intrinsically weak anion exchangeproperties. They are used in anion scavenging by analyzing test mixturescomprised of ˜5 different proteins in the presence of variousconcentrations of SDS. Polylysine surfaces may not provide the exchangecapacity or strength needed to scavenge SDS from solution at relevantconcentrations (10's of millimolar). Diethylaminoethyl (DEAE) exchangegroups may be used as a replacement for the polylysine. Threederivatization schemes creating DEAE are possible. The first possibilityis to link diethylaminoethylamine to 500 kDa CMD using EDC-mediatedchemistry or CDI-activation. A necessary limitation to this approach isneed for complete saturation of the carboxylic acid groups with DEAE. Ifall groups are not converted to DEAE, the surface will have(unpredictable) characteristics of both the cation and anionfunctionalities. A second method will be to activate polylysine surfacesusing 1,5-difluoro-2,4-dinitrobenzene and link the DEAE to the surfacethrough the benzylhalide. This has been done successfully using the1,5-difluoro-2,4-dinitrobenzene method for selectively immobilizingpeptides via their N-terminus. A possible downfall of this method,however, is the potential of severely cross-linking polylysine duringthe activation step, essentially making the surface inert to furtherderivatization. A third chemistry is to link diethylaminoethylamine tosurfaces amplified with CDT-activated dextran. This method is avariation of the CDI-CMD derivatization, and should reduce heterogeneouscation/anion exchange properties, as well as have a significant exchangecapacity. All of these preparation methods have been tried and assayedusing test peptide mixtures in the presence of SDS.

Enzymes, Assays, Incubator Enzymes

Another embodiment uses combinations of biomolecules to accomplishcombined multifunctional operations. Combined profiling, for example,uses two enzymes, such as endoproteases in combination with alkalinephosphatase, capable of mapping and subsequent dephosphorylation ofbiomolecules. Such an application is of value when elucidating thespecific phosphorylation sites present in e.g., regulation proteins. Theimmobilization of the phosphatases with various endoproteases isaccomplished with the ultimate goal of generating surfaces capable ofpH-dependent operation. Activity can be gauged by assaying smallphosphapeptides available from commercial suppliers.

Yet another embodiment uses snake venom phosphodiesterase (PD). Throughpartial sequencing, targets successfully derivatized with PD will add anextra dimension of specificity of PCR-based primer extension methodsused in the analysis of short DNA fragments containing mutations presentin DNA micro-satellites. The immobilization of PD to bioreactive targetsmay be accomplished, by gauging activity using small oligonucleotides.Studies involving both AP and PD relies on the general derivatizationprotocols according to the present invention.

Incubator

A common occurrence during proteolytic digestion using bioreactivetargets in combination with the affinity microcolumns is the tendency ofthe sample to dry out (on the target or target array) during the courseof digestion. A modified oven/incubator capable of performing digestsover a range of temperatures (25-60° C.) has been constructed thatsurrounds the bioreactive targets in a constant humidity environment.Digests have been performed for times greater than one-hour using thisincubator with little loss in sample volume. The devices is small andportable (dimensions: 4″×6″×1″; wt. ˜0.5 lbs.; 120 V), operates at asingle temperature (40° C.—sufficient for most applications of thebioreactive array), capable of maintaining a sample volume of 1-2 μL fortimes as long as one-hour using “matched set” targets, and amenable touse with robotic stations.

Biomolecular Applications Example 18 Beta-2-Microglubulin (β₂M) Analysisfrom Urine

Porous glass molecular traps, manufactured according to example 1 above,were activated and derivatized in batches (30-50 per batch) prior topacking into the pipettor tips forming an affinity pipette. After acidconditioning (with 0.05 M HCl for 1-hour, air-dried), the porous glassmolecular traps were treated with 10% amino-propyl triethoxysilane(Aldrich, Milwaukee, Wis.) in anhydrous toluene for 12-under reflux. Theamine-functionalized porous glass molecular traps were then equilibratedin reaction buffer (100 mM sodium phosphate, pH 4.8, 100 mM NaCl) for 15minute in a reaction vessel under slight vacuum. After equilibration,the buffer was replaced with a mixture of 15 kDa molecular masscarboxylated dextran (CMD, Fluka, Milwaukee, Wis.) and1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC, Sigma, St. Louis,Mo.) (10 mg/mL each in the reaction buffer) and the air was againevacuated from the reaction vessel. The reaction was allowed to proceedfor 1 hour (with two subsequent additions of EDC to the reaction mixtureat ˜20 and 40 minutes into the reaction) before terminating and rinsing.Prior to coupling of the antibody, the CMD-amplified porous glassmolecular traps were rinsed vigorously with 100 mM sodium phosphate, pH8.0, 0.5 M NaCl. The porous glass molecular traps were then activatedfor 10-minutes with EDC/N-hydroxy succinimide (NHS, Sigma, St. Louis,Mo.) (100 mM each, in H20) and incubated with the affinity purifiedrabbit anti-human β₂m IgG (DAKO, Carpinteria, Calif.) (0.1 mg/mL, in 20mM sodium acetate, pH 4.7). Uncoupled antibody was removed by extensiverinsing with HBS buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005%Surfactant P20). This manufacturing process yielded affinity pipetteswith a binding capacity estimated at 10-100 pmol, while having a deadvolume of approximately 1.5 μL. The anti-β₂m affinity pipettes werefound to be stable and active for a period of at least three-monthsfollowing antibody immobilization (by storing at 4° C. in HBS buffer).

Biological Fluids

All fluids were obtained immediately prior to use; protease inhibitorcocktail (PIC, Protease Inhibitor Cocktail Set III, Calbiochem, LaJolla, Calif.) was added immediately in order to minimize possibleproteolytic degradation of β₂m.

Tears. Human tears were collected by washing the eye withdoubly-distilled water (ddH₂O) and collecting the rinse. A 20 μL aliquotof the eye rinse was mixed with 180 μL BBS buffer and used as stocktears solution. This stock was further diluted by a factor often witheither water, (for MALDI-TOF analysis) or HBS buffer (for MassSpectrometric Immuno-Assay (MSIA) analysis).

Plasma. 44.7 μL of human whole blood were collected under sterileconditions from a lancet-punctured finger using a heparinizedmicrocolumn (Drummond Scientific Co., Broomall, Pa.), mixed with 205 μLHBS buffer and centrifuged for 30 seconds (at 7,000×g) to pellet the redblood cells. A 50 μL aliquot of the supernatant was mixed with 200 μLHBS and the resulting solution was used for MSIA; an aliquot was furtherdiluted (10 fold) with ddH₂O for MALDI-TOF analysis.

Saliva. Human whole saliva was diluted by a factor of 100 in ddH₂O orHBS buffer in preparation for MALDI-TOF or MSIA, respectively.

Urine. Human urine was prepared for MALDI-TOF by a 100-fold dilutionwith ddH₂O; a two-fold dilution with HBS buffer was used for MSIA.

Mass Spectrometric Immunoassay (MSIA)

MSIA was performed on the biological fluids by repeatedly drawing afluid 20 times) through an anti-β₂m-affinity pipette using a hand-heldP-200 micropipettor. After the repetitive-flow incubation, the affinitypipette was rinsed with 2 mL of HBS buffer (by drawing the HBS throughin 200 μL aliquots and then discarding), followed by a 1 mL rinse withddH₂O (using the same wash and discard approach). At the final discardof the water rinse, it was checked that all residual water was expelledfrom the affinity pipette. The retained compounds were eluted from theaffinity pipette by drawing a 3 μL aliquot of matrix solution (saturatedsolution of α-cyano-4-hydroxycinnamic acid (ACCA; Aldrich, Milwaukee,Wis.) in 1:2, acetonitrile: ddH₂O, 0.2% TFA) into the affinity pipette(enough to cover the microcolumn), upon which the matrix/eluent mix wasdeposited directly onto a MALDI-TOF target. MALDI-TOF mass spectrometrywas performed using a mass spectrometer. Briefly, the instrument uses atwo-stage 30 kV (2×1 cm; 15 kV/stage) continuous-extraction source toaccelerate ions to the entrance of a 1.4 m flight tube containing an ionguide-wire. Ions generated using a pulsed N₂ laser (337 nm) weredetected using a hybrid single channel plate/discreet dynode multiplierbiased at −3.8 kV. Spectra were recorded using an averaging transientrecorder while monitoring individual laser shots using a separateoscilloscope and attenuating laser intensity (in real-time) duringacquisition. All spectra were acquired in the positive-ion mode.

Quantification

Internal reference. Equine β₂m (Eβ₂m) was chosen as an internalreference for quantification because of its high degree of similarity tohuman β₂m (Hβ₂m) (˜75% sequence homology), resolvable mass differencefrom Hβ₂m (MW_(Eβ2m)=11,402.9; MW_(Hβ2m=)11,729.7) and because it waseasily obtainable. Horse urine was collected fresh (at a local stable)and treated immediately with protease inhibitor cocktail. Low solubilitycompounds were removed from the urine by overnight refrigeration (at 4°C.) followed by centrifugation for 5 minutes at 5,000×g. The urine wasthen concentrated 20-fold over a 10-kDa MW cut-off filter, withrepetitive HBS and water rinses and with several filter exchanges (4filters/200 mL urine). Treatment of 200 mL fresh urine resulted in 10 mLof β₂m-enriched horse urine which served as stock internal referencesolution for ˜100 analyses.

Working curve. Quantification of Hβ₂m was performed. Briefly, standardswere prepared by step-wise dilution (i.e., ×0.8, 0.6, 0.4, 0.2 and 0.1,in HBS) of a 1.0 mg/L stock Hβ₂m solution to a concentration of 0.1mg/L; the 0.1 mg/L solution served as stock for an identical step-wisedilution covering the second decade in concentration (0.01-0.1 mg/L). Ablank solution containing no Hβ₂m was also prepared. The samples forMSIA were prepared by mixing 100 μL of each of the standards with 100 μLof stock horse urine and 200 μL of HBS buffer. MSIA was performed oneach sample as described above, resulting in the simultaneous extractionof both Eβ₂m and Hβ₂m. Ten 65-laser-shots MALDI-TOF spectra were takenfrom each sample, with each spectrum taken from a different location onthe target. Care was taken during data acquisition to maintain the ionsignals in the upper 50-80% of the y-axis range and to avoid drivingindividual laser shots into saturation. Spectra were normalized to theEβ₂m signal through baseline integration, and the integral of Hβ₂m wasdetermined. Integrals from the ten spectra taken for each calibrationstandard were averaged and the standard deviation calculated. Acalibration curve was constructed by plotting the average of thenormalized integrals for each standard versus the Hβ₂m concentration.

Screening. Urine samples were collected from individuals, treated withprotease inhibitor cocktail and cooled to 4° C. The urine samples werecentrifuged for 5 minutes (at 5000×g) immediately prior to analysis toremove any precipitated material. In preparation for MSIA, 100 μL ofeach urine sample was mixed with 100 μL of stock horse urine and 200 ofHBS. This treatment is identical to that used in preparation of theworking curve, with the exception of replacing the standard with thehuman urine sample. MSIA was performed as described in the working curvesection.

Affinity Pipette Evaluation/Biological Fluids Screening.

The affinity pipettes were evaluated by screening a number of easilyobtainable biological fluids. The intent of the screen was to gauge thedegree of non-specific binding encountered from each of the fluids andto briefly investigate alternative rinsing protocols that reducecontributions from non-specific binding. FIG. 2 a shows a MALDI-TOFspectrum of diluted human tear and a spectrum showing tear compoundsretained during MSIA. High-level proteins present in the tears dominatethe MALDI-TOF spectrum: lysozyme (MW_(calc)=14,696; MW_(obs)=14,691) andtear lipocalin (MW_(calc)=17,444; MW_(obs)=17,440). Other polypeptidesignals are observed in the 2-5 kDa range, as well as a low-intensitysignal at m/z=11,727 Da, presumably due to β₂m. The MSIA spectrum showssignals due to the selectively retained β₂m (MW_(calc) 11,729;MW_(obs)=11,731) and attenuated signals for the lysozyme and othernon-specified compounds. FIG. 2 b shows MALDI-TOF and MSIA spectra ofdiluted human plasma. As is commonly observed during direct analysis ofserum or plasma, the MALDI-TOF spectrum is dominated by signalsoriginating from albumin. Other lower m/z signals are also present;however, β₂m signals are not observed. The MSIA spectrum shows strongsignals due to the selectively retained β₂m and few other signals fromnon-specified compounds. FIG. 2 c shows spectra of diluted saliva(MALDI-TOF) and salivary proteins retained during MSIA. The MALDI-TOFspectrum shows a number of signals in the 1-18 kDa range, mostprominently in the peptide region; signals corresponding to β₂m are notobserved. The MSIA spectrum, obtained after using the normal rinseprotocols, shows signals due to the selectively retained β₂m and anabundance of non-specified compounds in the low molecular mass range. Asecond MSIA analysis was performed in which an additional rinse with0.05% sodium dodecylsulfate (SDS) was included between the BBS and theddH₂O rinses (FIG. 2 c). The SDS rinse, although not completelyeliminating the low mass signals, did significantly reduce theircontribution to the mass spectrum without a proportional reduction ofthe β₂m signal. FIG. 2 d shows spectra resulting from the analysis fhuman urine. The MALDI-TOF spectrum shows a number of signals in thepeptide region and an absence of signal for β₂m. The MSIA spectrum isdominated by signals from the β₂m, with few additional signals fromnon-specified compounds.

The porous glass molecular traps used in the affinity pipettes performedwell in the screening of the biological fluids. Intermediate CMDamplification of the amine functionalized porous glass molecular trapsprovided a largely hydrophilic surface with multiple attachment points(carboxylic acid groups) for coupling of the antibody. As a result, theantibody load of each affinity pipette is more than sufficient tocapture low levels of β₂m without saturation of the antibody. Also, thehydrophilic surface can be washed free of most non-specifically boundcompounds by rinsing with aqueous ionic buffers. With the exception ofthe saliva sample, MSIA exhibited reasonably clean mass spectra showingpredominantly signals derived from β₂m. The SDS wash of the salivascreen, although improving spectral quality, did not completelyeliminate all of the non-specified compounds. Upon closer investigationit is found that those compounds (identified by mass as lysozyme,α-defensins and histatins) have pIs of ˜10, suggesting retention viacharge interactions (with free-carboxyl groups) that are not broken bythe moderate pH (7.8) and salt (150 mM NaCl) content of the HBS buffer.Thus, other rinsing combinations (e.g., high-salt or differentdetergents) will need to be investigated if the salivary screen isdeemed to be of biological significance. It is worth noting, however,that the presence of the non-specified compounds (in any of the samples)did not interfere with the unambiguous determination β₂m, which wasidentified by virtue of direct detection at its characteristic molecularmass.

Quantification

Protein quantification using MALDI-TOF requires use of internalstandards to compensate for varying laser intensities and spot-to-spotdifferences in sample composition that give rise to fluctuations inanalyte ion signal. Although proteins with characteristics unlike thoseof the analyte may be used as internal standards (as has been shownduring protein quantification directly from mixtures or during MALDI-TOFquantification of affinity-retrieved species by addition of an internalreference standard to peptides eluted from beaded affinity media),internal reference standards that behave similarly to the analyte duringlaser desorption/ionization are generally preferred. This prerequisiteis met during MSIA by choosing internal references that share sequencehomology with the target protein: enzymatic/chemically-modified versionsof the targeted protein, truncated/extended recombinant forms of thetarget proteins, the (same) target protein recombinantly expressed inisotopically-enriched media (e.g., ¹⁵N or ¹⁸O) or the same protein froma different biological species. Given that the receptor is able tocapture both the target protein and the internal reference, MSIA can bedesigned around a single receptor system. Alternatively, a two-receptorsystem can be considered where one receptor is used to retrieve thetarget protein and a separate receptor is used to retrieve the internalreference.

The internal reference chosen herein was equine β₂m (Eβ₂m), whichshares˜75% homology with its human counterpart and is ˜300 Da lower inmass than Hβ₂m (thus, both species share similar characteristics and areeasily resolved in the mass spectra). Even though no data could be foundon the relative dissociation constants between the polyclonal anti-β₂mIgG and Hβ₂m or Eβ₂m, preliminary studies showed that the antibodyexhibited cross-reactivity sufficient to retain both species. FIG. 3 ashows spectra representing MSIA analyses of Hβ₂m standards in aconcentration range of 0.01-1.0 mg/L. Each spectrum, normalized to theEβ₂m signal, is one often 65-laser shots spectra taken for eachcalibration point. Plotting the average of the 10 normalized Hβ₂mintegrals for each standard versus the Hβ₂m concentration results in theworking curve shown in FIG. 3 b. Linear regression fitting of the datayields I_(Hβ2m)/I_(Eβ2m)=4.09[Hβ₂m in mg/L]+0.021 (R²=0.983), with aworking limit of detection at a S/N>3 of 0.0025 mg/L (210 pM) and alimit of quantification of 0.01 mg/L (850 pM). The standard error of allpoints of the working curve is ˜5%.

Quantitative Determination of β₂M in Urine Samples

Ten samples were collected from four individuals: female (31 years,pregnant; 1-sample (F31)), male (30 years; 4-samples over two days(M30)), male (36 years; 2-samples over two days (M36)) and male (44years; 3-samples over two days (M44)). All of the individuals were in astate of good health when the samples were collected. Results from MSIAof the ten urine samples are shown in FIG. 4. The bars depict the β₂mconcentration determined for each sample, while the inset spectra aboveeach bar show the respective Hβ₂m signals normalized to Eβ₂m. The datafor the ten samples show remarkable consistency, with an average β₂mconcentration of 0.100±0.021 mg/L (high=0.127 mg/L; low=0.058 mg/L). Anadditional analysis was performed on a urine sample obtained from an86-year old female (F86) who had recently suffered a renal infection.Because of the significantly higher level of β₂m found in this sample(see inset spectrum) it was necessary to quantitatively dilute the urineby a factor often in order to keep the β₂m signal inside the dynamicrange of the working curve and accurately establish the β₂mconcentration in F86 (at 3.23±0.02 mg/L).

Post Translational Modifications

The mass-selective detection of MSIA makes possible the discovery andquantification of variants of β₂m that may be present in urine. Duringquantitative screening of the urine samples, a second, higher molecularmass species (Δm=+161 Da) was co-extracted with the β₂m. The species ispresumably a glycosylated (one hexose) form of β₂m, and is observed mostprominently in F86. FIG. 5 shows an overlay of two MSIA spectra takenfrom the urine of F86 (diluted×20) and M36 (no dilution; given forcomparison). The level of glycosylated β₂m is much greater in F86 thanin M36. The specific cause of the elevated level of the glyco-β₂m is atpresent uncertain.

Previous work has reported the quantification of glycosylated Hβ₂m bydirect MALDI-TOF analysis of serum into which glycosylated Hβ₂m wasdoped at reasonably high concentrations. Due to poor mass spectralresolution and interferences from the serum, a curve-fitting routine wasused to deconvolute signals from multiple Hβ₂m glycoforms, the integralsof which were then normalized to wild type β₂m for use in constructing aworking curve of reasonable linearity (R²=0.88). Although the datapresented here is significantly better and clearly able to supportrigorous quantification without aid of fitting routines, it is uncertainwhether the working curve constructed for the unmodified β₂m can be useddirectly for the rigorous quantification of the glycosylated β₂m. Suchcorrelation requires that the affinity constants for, and thedesorption/ionization efficiency of, both β₂m forms are equal. Regardingthe affinity constants, the affinity-purified polyclonal antibody usedherein clearly shows broad cross-reactivity (as demonstrated by theco-extraction of Hβ₂m and Eβ₂m) so it is highly probable that both Hβ₂m.forms are extracted with similar efficiencies. Similarly, the additionof a single carbohydrate moiety should not severely attenuate therelative desorption/ionization efficiency. As a result, theconcentration of the glycosylated β₂m form might be estimated using theβ₂m-working curve at 0.072 mg/L—roughly the same concentration as wildtype β₂m in the urine samples from the healthy individuals.

Regardless of whether both wild type and glycosylated β₂m can bequantified using a single working curve, it is important to note thatthe concentration of the wild type β₂m determined during MSIA doesaccurately reflect the concentration of only the wild type β₂m and notthe combination of both of the species. Thus, MSIA holds a particularadvantage over other techniques that are unable to differentiate betweensimilar forms of a target analyte. In that elevated β₂m levels are usedas a general indicator of immune system activity, whileβ₂m-glycosylation has been associated with more specific ailments (e.g.advanced glycosylated end-products associated with dialysis relatedamyloidosis), MSIA is able to deconvolute these independent contributingfactors and yield results that more accurately connect a specificbiomarker with a specific ailment.

The manufacturing process used in this report yielded affinity pipetteswith an estimated binding capacity of 10-100 pmol, while having a deadvolume of approximately 1.5 μL. This binding/elution ratio was found toadequately match the β₂m concentrations found in the biological fluids.Moreover, the efficient capture of β₂m using the affinity pipettes keptthe sampling volume low (less than 100 μL of biological fluid). Inaddition, the affinity pipette chemistries employed herein exhibitedlittle non-specific binding for three of the four biological fluids, andeven in the fourth (saliva) did not introduce analytical interferencesinto the analysis.

The quantitative capabilities of MSIA are clearly demonstrated in thepresent invention. The β₂m concentration range investigated herein(0.010-1.0 mg/L) is adequate to cover the β₂m levels in all fluids. Goodlinearity is observed over the two decade ranges (R²=0.983) with anoverall error of ˜5%. Important to accurate quantification is the choiceof an appropriate reference standard, which in this example wasfulfilled by use of horse urine enriched in β₂m. However, even thoughthe horse urine is viewed as an ideal background media (because it moreclosely mimics the true analytical media than buffers), it will need tobe replaced in future analyses with purified Eβ₂m in order to ensureconsistency when analyzing a large number of samples over long periodsof time.

Although the screening study presented here was not extensive enough tobe considered a clinical study, it does demonstrate the utility of MSIAin accurately identifying and quantifying β₂m directly in biologicalsamples, such as urine samples. The four baseline individualscontributing urine to the project were considered healthy, not sufferingfrom any known genetic afflictions linked to β₂m or having suffered fromany ailments in the month immediately preceding the analyses.Qualitative evaluation of the β₂m retained from the samples revealedsingle signals for β₂m with molecular mass corresponding to thewild-type sequence of the protein (within 0.02% experimental error;spectra internally calibrated by using the Eβ₂m signals; see FIG. 4).Quantitative analysis within the group showed remarkably constant β₂mlevels that are consistent with those found in control groups duringother studies. By contrast, the urine sample obtained from an olderindividual of poor health showed a marked increase in urinary β₂m level(˜30-times greater). It should be noted that this estimate was madewithout interference from a higher-mass variant of β₂m, which wasreadily detectable in the mass spectra (see FIG. 5). The most reasonableexplanation for the observation of two mass-shifted (yet related)signals in the mass spectrum is the presence of: 1) the wild-typeprotein, and 2) a variant existing due to either a genetic polymorphismor a posttranslational modification. In this particular case, thevariant is most easily identified by the mass shift of +161 Da as aglycosylated form of β₂m; variants due to genetic polymorphisms areessentially ruled out because the mass shift is greater than thatresulting from any single nucleotide polymorphism (i.e., [TGG]−[GGG];resulting in Trp−Gly; dm=129.15 Da). However, had variants possessing asignificant mass shift (>15 Da) as a result of a genetic polymorphismbeen present in the sample, they would have been as readily recognizedas the glycosylated β₂m.

Lastly, in that MSIA analyses are fairly rapid and relatively easy toperform, the approach lends itself particularly well to the rapiddevelopment of analytical methods and the analysis of large numbers ofsamples. The rapid rate of analysis opens the possibility of real-timemethod development in which changes in incubation and rinse protocolscan be readily implemented when the results from a just completed trialare analyzed. Once developed and optimized, these methods could bereadily applied to screening of biological fluids. Herein the sampleswere analyzed at a rate of ˜3/hour, which allowed for several analysesto be performed on a given individual within a single day (as shown inFIG. 4, M30). This rate of analysis was essentially limited byinstrumentation that is not designed to accommodate multiplesamples—each analysis herein (preparation-through-analysis) wasperformed individually. However, it is feasible to increase this rate ofanalysis to hundreds-per-day by use of parallel pipetting stations andmass spectrometers that accept multiple samples on an arrayed format. Inthis manner, pipetting stations addressing 96-well format titer plateswill now be described to simultaneously prepare, modify, incubate,capture and rinse the multiple samples using multiplexed affinitypipettes. The samples are then eluted onto a mass spectrometer targetarray of the same format.

Robotic Integration

The mass assay system, FIG. 7 a, is a system for high throughput nascentbiological fluid analyte extraction by affinity microcolumns within aconduit system comprising one or more functional pre-, use, analysis,and post-stations. The pre-stations initiate sample information,organize, prepare and formulate components, sample arrays and targetsfor primary and analysis workstation use. Sample identification,performed on incoming biological fluids, which ascertains initial sampleparameters, such as bio-sample type (blood, urine, cell culture media,etc.), patient history, disease state, chemical parameter profile (pH,turbidity, etc.), and the like, results in a sample classificationdatabase for integration with final output analysis data promotingcongruent and feedback databases. Pre-station high performance fluidmanipulation, with accompanying labeling and tracking, allows fordistribution of identified biological fluid from sample source intocompartmentalized individual sampling array, labeled (e.g.,bar-code/laser reader) for tracking, with further inline manipulation,such as appropriate dilution or modification (pH, surfactant addition,etc.). Another function of the pre-station is formulation and loading ofthe array components. Here, component arrays, including, but not limitedto, solid components (plastics, glasses, metals and the like), fluidreagents, targets and the like, are formed and/or distributed intoappropriate arrays and loaded into the primary workstation. From theprestation, the array of molecular traps are accessed by aninitiation/reservoir/sample station, the primary station, or relocatedto a use station for sample processing.

Example processes performed in the prestation include, but are notlimited to, assembly of porous molecular traps into affinity pipettes,array labeling, sample preparation, sample modification. These processesare carried out using solid sample array(s) (plastic microtiter plates,etc.), array labels, chemical/modifying reagent (e.g. storage reagents,activation reagents, buffers, surfactants, reducing reagents, etc.),affinity reagents and ligands (biological mimics, antibody(s),antigen(s), etc.), rinse fluids (buffers, ultra pure water, etc.),desorption fluids (MALDI matrix, acids, bases, surfactants, etc.) andanalysis target(s).

The pre station combines ligand and analyte in separate microcolumnactivation, sample retrieval/separation and sample transfer/elutionfunctions. Preferably, multiple samples are loaded into the pre stationand spatially arranged in an array commensurate with the array ofmolecular traps with one sample for each molecular trap in the array ofmolecular traps.

From the pre station, the array of samples is automatically relocated toa use station. The use station is where the sample, and specificanalytes contained therein are processed. In one embodiment, one end ofthe array of molecular traps is lowered into the sample and the samesample is drawn into each molecular trap. Since each molecular trap hasaffinity receptors located on surfaces of the molecular trap, drawingthe sample into the molecular trap contacts any specific analyte soughtafter with the affinity receptors. In the array, each molecular trap mayhave different affinity receptors from that of other molecular traps inthe array, thereby enabling the targeting of different specific analytesfrom the same or different media. Sample material may be drawn into themolecular trap singly or multiple times. After sufficient specificanalyte has been captured by the molecular traps in the array, residual,or non-captured, media are washed away with at least one rinse.(However, other embodiments may not require a rinse step.) Afternon-targeted compounds have been washed away, captured specific analyteare eluted from the molecular traps by contacting them with a solutionselected to interrupt the affinity interaction. The eluted specificanalytes are then either prepared directly for mass spectrometry or forfurther processing, e.g., enzymatical/chemical modification, followed bysubsequent preparation for mass spectrometry. The pre stations maycomprise multiple positions for chemical modification, molecular trapfunctionalization, biological fluids analysis and/or transfer. Finally,the eluted specific analytes are relocated to a target array by stampingthem onto the target array.

With reference to FIG. 7 b, a preferred embodiment of the use stationfurther comprises a microcolumn manifold to which the array of moleculartraps are attached. The microcolumn manifold attaches to a robotic headthat then physically moves the array of molecular traps between eachprocessing station. This physical movement of the microcolumn manifoldmay be in a rectangular (xy) or circular (carousel) manner.Alternatively, the microcolumn manifold in the use station may bestationary and the processing stations relocated under the array ofmolecular traps. Like the physical movement of the microcolumn arraydescribed above, the physical movement of the processing stations may bein a rectangular (xy) or circular (carousel) manner. A furtherembodiment contemplates the physical movement of both the microcolumnmanifold and the processing stations together.

In a preferred embodiment, the target array is automatically relocatedto a storing/loading station that is capable of containing at least onetarget array. From the storing/loading station, the target array istransferred into an automated mass spectrometer capable of multi-sampleinput and automatic processing/data analysis using an interactivedatabase.

Automated mass spectrometry is performed with either the specificanalyte or modified fragments detected with high precision. Softwarecapable of recognizing differences between samples, or from a standard,is used to aid in the analysis of large numbers of samples and generateproprietary databases from which to establish novel information systemsi.e., structure function systems, clinical systems, diagnostic systemsand biochemical systems. Biochemical systems generally refer to achemical interaction that involves molecules of the type generally foundwithin living organisms, including the full range of catabolic andanabolic reactions which occur in living systems such as enzymatic,binding, signaling and other reactions. Other, biochemical systems,includes model systems that are mimetic of a particular biochemicalinteraction. Examples demonstrated within the context of this presentinvention or of interest in practicing the present invention include,e.g., receptor-ligand interactions, protein-protein interactions,enzyme-substrate interactions, cellular signaling pathways, transportreactions, genotyping and phenotyping.

After analysis by mass spectrometry, the target array may be transferredto a post-station for sample processing or additional analysissubsequent to the mass spectrometric analysis.

Accordingly, in one aspect, the present invention will be useful inscreening for ligands or compounds that affect an interaction between areceptor molecule and its ligand.

In-Robot Functionality Example 19 In-Robot AmineActivation/Derivatization of Functionalized Porous Molecular Traps

Another approach involves robotic integration of the previouslymentioned formats during which such protocols as glutaraldehyeactivation and ligand coupling, are used in a concerted in-robotactivation/derivatization. Here, amine functionalized porous glassmolecular traps are dry loaded/seated (either manually or machineassisted) into warm p-200 wide bore pipette affinity pipettes (RobbinsCorp.) and loaded into a plastic 96 rack. This rack is integrated ontostation one of a six-staged robot, loaded with microtiter platescontaining various coupling and rinsing solutions, that is linked to apersonal computer and software controlled. Glutaraldehye coupling toamine microcolumns occurs via a first 96 well microtiter platecontaining 110 μL per well of 25% glutaraldehyde solution in 0.10 Msodium phosphate buffer, pH 7.8, stationed at position two, usingone-hundred aspiration repetitions. This initial coupling step requiresextensive HBS buffer rinses (110 μL/well) attained at station three(containing a second 96 well microtiter plate, fifty aspirationrepetitions), resulting in a rapid in-robot mediated activation coupling(reaction time 10-20 minutes). After a final water rinse at station four(˜fifty aspiration repetitions), the activated aldehyde groups on theglutaraldehye matrix are incubated with the protein antibody of interestat station five (0.1-1 mg/mL, 55 μL/well, 200 aspirations). Uncoupled(excess) antibody is removed by extensive rinsing at station six withHBS buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20,fifty aspiration repetitions).

Example 20 In-Robot Amine Activation, Amplification, Reactivation andDerivatization of Amine Microcolumns

An extension of the approach demonstrated above is robotic integrationof glutaraldehye activation with subsequent in-robot amplification withrecurring activation prior to ligand coupling, as a concerted in-robotactivation/amplification/derivatization. In this example, aldehydefunctionalized porous glass molecular traps (aldehyde microcolumns) areprepared and loaded into positioned one. Then the activated aldehydegroups on the glutaraldehye microcolumns are incubated with polylysine(30-300 kDa) loaded at position two (110 μL, 1 mg/mL in HBS) of therobot stage, resulting in formation of a polymeric scaffold from whichto conduct another round of glutaraldehyde mediated activation andantibody conjugation to the amplified micro-top surface.

This initial coupling step is followed by extensive rinses at stationthree, fifty aspiration repetitions of HBS buffer, resulting in a rapidin-robot mediated activation coupling (reaction time half an hour).After a final water rinse at station four (˜fifty aspirationrepetitions), the activated aldehyde groups on the glutaraldehye matrixare incubated with the protein antibody of interest at station five(0.1-1 mg/mL, 55 μL/well, 200 aspiration repetitions) after which theMASSAY microcolumns become affinity pipettes. Uncoupled (excess)antibody is removed by extensive rinsing at station six with HBS buffer(10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20).

Example 21 MALDI-TOF Chemically Masked Target Preparation forMicrocolumn Elution

Arrayed targets present hydrophilic targets that function to localizeanalyte samples delivered from microcolumns under eluting conditions.These elution conditions vary depending upon the analysis intent,ranging from the general use of MALDI-TOF matrix (ca., alphacyano-4-hydroxy cinnamic acid in 1:2 ratio of acetonitrile: 0.2% TFA inwater), dilute acids, dilute bases, caotropic agents and the like.

In the present example, the target surface consists of a contrast arraywhere a hydrophillic target functions in concert with a hydrophobicbackground in a chemical mask prepared for a directed analysis,depending upon the type of biomedia used in the assay of interest.Regardless of the intent, the first requirement is cleaning of thesurface in a series of rinses and incubations. Initially the targetsurface is cleaned with detergent, water rinsed, methanol rinsed, andthen incubated with 10-15% hydrogen peroxide solution in water at roomtemperature (or elevated temperatures) for thirty minutes to one hour,After which the cleaned surface is rinsed in water, methanol, nitrogendried and the target areas derivatized with the alkyl mercaptan ofinterest. For a cation exchange surface 11-mercaptoundecanoic or3-mercapto-1-propanesulfonic acid is used as a saturated solutionprepared in an organic solvent (ca., isopropanol). The surface is eithercoated with a saturated solution of octadecyl mercaptan immediately orrinsed with isopropanol followed by methanol and subsequently coatedwith a saturated solution of octadecyl mercaptan. A chemicallycontrasted, or masked, surface results for microcolumn analytelocalization and sample analysis by mass spectrometry. These cationexchange surfaces are designed for working in a general biological mediaand are particularly useful when assaying out of urine. Alternatively,masked targets are generated with positively charged groups for use asanion exchangers.

Example 22 Integrated High Throughput System MALDI-TOF Analysis ofBiological Samples

Integrated system parallel processing and analysis of biologicallyrelevant biomolecules out of nascent biological fluids is illustrated inthis example demonstrating the capabilities of the high-throughputsystem. Demonstrated in FIG. 8 is the high-throughput semi-quantitativeanalysis of beta-2-microglobulin (β₂m) from human plasma samplesperformed using the integrated system and methods of the presentinvention. Aliquots of diluted (5 fold) human plasma samples collectedfrom six individuals were prepared for parallel screening on a 96-wellsample plate. Each well received a 15 μL plasma aliquot (the samplesfrom the six individuals were randomized on the 96-well plate), 7.5 μLof equine plasma (undiluted, containing equine β₂m,MW_(eq.β2m)=11,396.6, MW_(hum.β2m)=11,729.2) and 128 μL of HBS (0.01HEPES, pH 7.4, 0.15 M NaCl, 0.005% (v/v) polysorbate 20, 3 mM EDTA)buffer. Eight of the 96 samples were chosen at random and 0.5 μL of 10⁻²mg/mL solution of β₂m was added to four of them and 1 mL of the same β₂msolution to the other four wells. Parallel sample processing entailedsimultaneous incubation/capture of the 96 samples on 96 anti-β₂mderivatized microcolumns. The polyclonal anti-β₂m microcolumns were madevia carboxymethyl dextran (CMD)-EDC mediated coupling of the antibody toamino-coated/modified microcolumns. Captured proteins were eluted fromthe microcolumns with a small volume of MALDI matrix (saturated aqueoussolution of α-cyano-4-hydroxycinnamic acid (ACCA), in 33% (v/v)acetonitrile, 0.2% (v/v) trifluoroacetic acid) and stamped onto a MALDItarget array surface comprised of self-assembled monolayers (SAM)chemically masked to make hydrophilic/hydrophobic contrast targetarrays. Each sample spot on the target array was analyzed using massspectrometry and the relative β₂m abundance determined by an automatedMALDI-TOF mass spectrometric analysis software routine. The mass spectraresulting from the high-throughput analysis of the 96 samples are shownin FIG. 8. Spectra taken from the samples that had the β₂m standardsolution added are shaded.

FIG. 9 bar graph visualization performs interpretation of thehigh-throughput system generated semi-quantitative data shown in FIG. 8.Each spectrum shown in FIG. 8 was normalized to the equine β₂m signalthrough baseline integration, and the normalized integral for the humanβ₂m signal determined. All β₂m integrals from spectra obtained fromsamples from the same individual were averaged and the standarddeviation calculated. In the same way, the integrals for the samplesspiked with 0.5 and 1.0 μL solution of 10⁻² mg/mL β₂m were calculatedand averaged. Plotted in this figure are the average values of thenormalized human β₂m integrals for the samples from the six individualsand the spiked samples. The bar graph clearly establishes increased β₂mlevels in the spiked samples, illustrating the value of thehigh-throughput semi-quantitative analysis performed with the system andmethods described in this invention in establishing increased β₂m levelsin human blood that are associated with various disease states.

Another demonstration of the integrated system and methods describedwithin the present invention comes from FIG. 10 high-throughputquantitative analysis of β₂m from human blood. The plasma samples fromsix individuals were prepared as described in FIG. 8. Eighty-eight wellsof the 96-well sample plate received 15 μL plasma aliquots (the samplesfrom the six individuals were randomized on the 96-well plate), 7.5 μLof equine plasma (undiluted) and 128 μL of HBS buffer. A series ofdilutions of a 7.6×10⁻⁴ mg/mL standard solution of purified human β₂mwere prepared (spanning a concentration range of 7.6×10⁻⁴ to 1.14×10⁻⁴mg/mL) and used as samples (15 μL of each) in the last column (8 wells)on the 96-well plate. Parallel sampling processing and MALDI-TOF MSanalysis was performed as described for FIG. 8, using the polyclonalanti-β₂m microcolumns. The mass spectra resulting from thehigh-throughput analysis of the 88 samples and the 8 standards are shownin this figure. Spectra taken from the standard samples are shaded.

FIG. 11 b is the calibration curve constructed from the FIG. 11 a datafor the standard samples shown in FIG. 10. The calibration curve ispresented for the purpose of determining the β₂m concentrations in thehuman plasma samples screened via the high-throughput analysis using theintegrated system and methods described in the present invention.Representative spectra of the data for each standard used to generatethe working curve are presented overlain in FIG. 11 a. Each spectrum wasnormalized to the equine β₂m signal through baseline integration, andnormalized integrals for the human β₂m signals determined. Integralsfrom five spectra taken for each calibration standard were averaged andthe standard deviation calculated. A calibration (standard) curve wasconstructed by plotting the average of the normalized integrals for eachstandard vs. the human β₂m concentration in the standard sample(adjusted for the human plasma dilution factor). The working curvegenerated is shown in FIG. 11 b. The concentration range was spannedwith good linearity (R²=0.999) with overall standard deviation of theline of <2%.

FIG. 12 is the bar analysis of the data shown in FIG. 10 using thestandard curve constructed in FIG. 11 b is illustrated. Each spectrumfor the 88 samples from FIG. 10 was normalized to the equine β₂m signalthrough baseline integration, and the normalized integral for the humanβ₂m signal determined. The human β₂m integrals for the same individualwere averaged and the standard deviation calculated. The values of theaveraged integrals were substituted in the equation derived from thestandard curve and the concentration of human β₂m was calculated foreach individual. The range of concentrations determined was from 0.75 to1.25 mg/L.

Example 23 Integrated Combined System Approach Incorporating HighThroughput Affinity Retrieval With Bioreactive Array MALDI-TOF Analysisfor Point Mutations

FIG. 13 is the qualitative high-throughput screening of transthyretin(TTR) for posttranslational modification (PTM) and point mutations (PM)was performed using the integrated system and methods described herein.Aliquots of diluted (5-fold) human plasma samples collected from sixindividuals were prepared for parallel screening on a 96-well plate.Each well received a 15 μL plasma aliquot (the samples from the sixindividuals were randomized on the 96-well plate), and 135 μL of BBSbuffer. Parallel sampling processing entailed simultaneousincubation/capture of the 96 samples on 96 anti-TTR derivatizedmicrocolumns. The polyclonal anti-TTR microcolumns were made viaglutaraldehyde-mediated coupling of the antibodies toamino-coated/modified microcolumns. Captured proteins were eluted fromthe microcolumn array with a small volume of MALDI matrix (saturatedACCA solution) and stamped onto a MALDI target array surface comprisedof self-assembled monolayers (SAM) chemically masked to makehydrophilic/hydrophobic contrast targets. Each sample spot on the targetarray was analyzed using mass spectrometry and the relative TTRabundance determined by an automated MALDI-TOF mass spectrometricanalysis software routine. The mass spectra resulting from thehigh-throughput analysis of the 96 samples are shown in FIG. 13. In allof the spectra, the TTR signal is accompanied by another signal athigher mass, indicating posttranslationally processed TTR form. Inaddition, all spectra resulting from the analysis of one plasma sampleshowed two additional signals at masses ˜30 Da higher than the two“original” TTR signal. See FIGS. 15 and 16 for identification of thesepeaks.

FIG. 14 is the identification of the posttranslational modifications andpoint mutations observed in the high-throughput TTR analysis wasperformed using the integrated system and methods described herein.Shown are representative spectra resulting from analysis of samples fromtwo individuals, showing the existence of two and four TTR signals,respectively. In the upper spectrum, two signals attributable to TTR areobserved. The signals correspond well to the theoretically calculatedmass of TTR (MW_(TTR)=13,762) and that of an oxidized TTR variant(TTR_(ox)) resulting from cysteinylation at Cys10 (introducing a massshift of +119 Da). In the lower spectrum, in addition to theabove-mentioned two TTR signals, two additional peaks at masses ˜30 Dahigher than the two “original” TTR signal are observed. See FIGS. 15 and16 for identification of these two peaks.

Continued analysis of the TTR point mutation, displayed in FIG. 15, isillustrated using combined high-throughput affinity retrieval in concertwith derivatized mass spectrometer target array in the system platformand the methods described in the present invention. The samples usedwere the same ones utilized for FIG. 14. TTR from diluted (50-fold, inHBS) human plasma was captured via polyclonal anti-TTR microcolumns, asdescribed for FIG. 13. Instead of matrix elution, the captured proteinswere eluted with a small volume of 10 mM HCl onto trypsin-conjugatedtargets containing buffered target spots (50 mM TRIS buffer pH 9.5) forsample pH modulation (buffer exchange). Shown in this figure are massspectra resulting from a twenty-minute trypsin digest done at 40° C. ofthe proteins eluted from the anti-TTR microcolumns. The resulting twotryptic peptide maps localize the mutation in the tryptic fragment-12(T₁₂), containing residues 104-127. A database search points to twopossible TTR mutations in this region of the sequence: Ala109→Thr [DNAbase change GCC→ACC], Δm=30.011 Da, and Thr 119-Met [DNA base changeACG→ATG], Δm=29.992 Da. The identification of the correct mutation isshown in FIG. 16.

FIG. 16 is the high-resolution reflectron mass spectrometry used indetermining the identity of the point mutations detected in the analysisof the plasma samples shown in FIG. 15 forming an integral part of theintegrated system and methods described in the present invention. Themonoisotopic signal for the tryptic digest fragment T₁₂(104-127) innormal (native) TTR shows at m/z=2644.922, denoting Δm=29.988 Dadifference with the monoisotopic signal for the mutant TTR. Accordingly,the point mutations is assigned to Thr119→Met, Δm=29.992 Da. This TTRpoint mutation results in a so-called “Chicago prealbumin” variant, anon-amyloid mutation. The results shown in FIGS. 13, 14, 15, and 16 incombination illustrate the use of the system and the methods describedherein in identifying posttranslational modifications and pointmutations via concerted high-throughput screening analyses of biologicalsamples.

Example 24 Integrated Combined System Approach Incorporating HighThroughput Affinity Retrieval With Bioreactive Array MALDI-TOF forAnalysis of Posttranslational Modification (PTM)

FIG. 17 a is representative of the qualitative high-throughput screeningfor posttranslational modifications present in biological fluidperformed using the integrated system and methods described herein.Concerted biological fluid phosphate analysis was performed usingchelator affinity pipettes in conjunction with alkaline phosphatase (AP)functionalized target array. Here, chelator affinity pipettes wereprepared as previously disclosed in accordance with example 14. Humanwhole saliva centrifuged and diluted 10 fold was either analyzed insample or after incubation with metal chelator affinity pipettes.Captured analyte from metal chelator affinity pipettes was then elutedusing dilute acid addition to disrupt the chelator/metal/analyteinteraction and stamped onto a hydrophilic/hydrophobic contrast targetarray or alkaline phosphatase functionalized target array. The laterincorporated buffer exchange for subsequent phosphate digests. Directanalysis of dilute human saliva significantly lacks proline richprotein-1 (PRP-1), the serine modified phosphate rich protein ofinterest. The FIG. 17 b(2) spectrum denotes metal chelator affinitypipette capture shown as two phosphate rich proteins, PRP-1 and PRP-3.The dephosphorylation mass signature is evident in spectral trace FIG.17 b(3) and complete in FIG. 17 b(4). Illustrating multi-analytedetection accompanied by partial and complete dephosphorylation ofphospho-proteins captured/digested out of biological fluid forpost-translational analysis, such as phosphorylation events.

Example 25 Integrated System Approach for Affinity Multi-ProteinInteractions Integral in Nascentprotein Complex Retrieval with MALDI-TOFAnalysis

Illustrative in this example is the interaction within proteins thatallow for retrieval of the native multi-protein complex present inbiological media or biological samples. Demonstrated in FIG. 18 is themulti-protein complex between retinol binding protein (RBP) andtransthyretin (TTR) from human plasma samples performed using theintegrated system and methods of the present invention. Polyclonalanti-RBP affinity pipettes were formed using glutaraldehyde mediatedamine base support surface coupling. Human plasma was prepared and usedas previously described in above examples. MSIA delineates in vivoaffinity retrieval of RBP and complexed TTR. The multi-protein complexbetween retinol binding protein (RBP) and transthyretin (TTR)illustrates protein interactions exiting in native protein complexes.

Example 26 Integrated System Approach Incorporating High ThroughputMulti-Analyte Retrieval and MALDI-TOF Analysis

Multi-receptor affinity microcolumn high-throughput approaches aredescribed in this example to illustrate multi-analyte analysis fromhuman plasma samples performed for rapid qualification andsemi-quantitation using the integrated system and methods of the presentinvention. FIG. 19 shows simultaneous rapid monitoring of multi-analytesfor relative abundance. Amine activated, polyclonal anti-b2m/CysC/TTRaffinity pipettes were used to rapidly capture their respective analytesout of human plasma as previously described. This then illustrates oneof the uses for multi-antibody affinity pipettes to β₂m, CysC and TTR torapidly monitor for biological fluid level modulation and to quantify amodulated protein event from their normalized relative abundance.Further illustrating one of the uses of affinity pipettes in theintegrated system and methods of the present invention for monitoringpotential β₂ m/CysC levels in acute phase of viral infection (ca. AIDS)or fibril formation from β₂m or TTR.

Shown in FIG. 20 is the rapid monitoring of extended multi-analyteaffinity pipettes in the integrated system and methods of the presentinvention. Combinations as well as individual polyclonal antibodyaffinity pipettes incorporating β₂m, TTR, RBP, Cystatin C or CRP wereused to capture respective analytes from human plasma (attained aspreviously stated in earlier examples). Illustrating another use formulti/single-antibody affinity pipettes to β₂m, CysC, TTR or CRP torapidly monitor for biological fluid level modulation and to potentiallyquantify a modulated protein event from their normalized relativeabundance. And further illustrates another of the uses of affinitypipettes for monitoring potential β₂m/CysC levels in acute phase ofviral infection (ca. AIDS) or fibril formation from β₂m or TTR.

Given the ability to increase sample throughput, the integration of ahigh throughput system for mass spectrometric analysis of biomoleculesfinds increased use in large-scale clinical, diagnostic, and therapeuticefficacy, applications where exceptional qualitative and quantitativeaccuracy are both needed in biologically important biomolecule analysisout of biological fluids.

As used herein, “affinity microcolumn” refers to a molecular trapcontained within a housing.

As used herein, “affinity reagent” refers to a chemical species on thesurface of the molecular trap and is chemically activated, oractivatable, and to which affinity receptors may be covalently linked.

As used herein, “affinity receptors” refers to atomic or molecularspecies having an affinity towards analytes present in biological mediaor biological samples. Affinity receptors may be organic, inorganic orbiological by nature, and can exhibit broad (targeting numerousanalytes) to narrow (target a single analyte) specificity. Examples ofaffinity receptors include, but are not limited to, antibodies, antibodyfragments, synthetic paratopes, peptides, polypeptides, enzymes,proteins, multi-subunit protein receptors, mimics, organic molecules,polymers, inorganic molecules, chelators, nucleic acids, aptamers, aswell as the below stated receptors.

As used herein, “analyte” refers to molecules of interest present in abiological sample. Analytes may be, but are not limited to, nucleicacids (DNA, RNA), peptides, hormonal peptides, hormones, polypeptides,proteins, protein complexes, carbohydrates or small inorganic or organicmolecules having biological function. Analytes may naturally containsequences, motifs or groups recognized by the affinity receptors or mayhave these recognition moieties introduced into them via processing suchas cellular, extracellular, enzymatic, chemical, and the like.

As used herein, “biological media” or “biological sample” refers to afluid or extract having a biological origin. Biological media may be,but are not limited to, cell extracts, nuclear extracts, cell lysatesand excretions, blood, sera, plasma, urine, sputum, sinovial fluid,cerebral-spinal fluid, tears, feces, saliva, membrane extracts, and thelike.

As used herein “chemically activate” refers to the process of exposingthe affinity reagent to chemicals (or light) in order to subsequentlyattach (or photoactivate) tethering linkers and affinity receptors.Compounds able to activate affinity reagents may be, but are not limitedto organic, inorganic, or biological reagents. Often, it is advantageousto activate the affinity reagent using multiple steps including the useof a tethering linker. As used herein, “tethering linker” refers tocompounds intermediate to the affinity reagent and the affinity receptorthat exhibit the desirable characteristics of being able to bederivatized with high densities of affinity receptor and showing lowbinding of non-specified compounds. The tethering linker may beintrinsically active or require activation for attachment. Suitabletethering compounds include but are not limited to homo/heterofunctional organics, natural and synthetic polymers and biopolymers.

As used herein, “dead volume” refers to the void volume within themolecular trap. As used herein, “low dead volume” refers to the range of1 nanoliter to 1 milliliter.

As used herein, “high flow properties” refers to a minimum flow rate of1 microliter per minute. Higher flow rates are considered to fall withinthe definition of high flow properties.

As used herein, “mass spectrometer” or refers to a device able tovolatilize/ionize analytes to form vapor-phase ions and determine theirabsolute or relative molecular masses. Suitable forms ofvolatilization/ionization are laser/light, thermal, electrical,atomized/sprayed and the like or combinations thereof. Suitable forms ofmass spectrometry include, but are not limited to, Matrix Assisted LaserDesorption/Time of Flight Mass Spectrometry (MALDI-TOF MS), electrospray(or nanospray) ionization (ESI) mass spectrometry, quadruple ion-trap,Fourier transform ion cyclotron resonance (FT-ICR), magnetic sector, orthe like or combinations thereof mass analyzers.

As used herein, “mass spectrometer target” or “mass spectrometer targetarray” refers to an apparatus onto or into which one or more analytesare deposited for subsequent mass spectrometric analysis. Generally,targets will accommodate numerous samples and are of various geometricalconfigurations depending on the nature of the mass spectrometer forwhich they are designed. Suitable materials for constructing targetsinclude metals, glasses, plastics, polymers, composites, and the like orcombinations thereof.

As used herein “molecular trap” refers to a high surface area materialhaving high flow properties and a low dead-volume, with affinityreagents bound to surfaces contained therein. The composition of thehigh surface area material may be, but is not limited to, crystal,glasses, plastics, polymers, metals or any combination of thesematerials. For example, these glasses may be silica glasses,borosilicates, sodium borosilicates, and other useful materials.

As used herein, the term “receptor” generally refers to one member of apair of compounds that specifically recognize and bind to each other.The other member of the pair is termed a “ligand” and includes suchthings as complexes, protein-protein interactions, multianalyteanalyses, and the like. Receptor/ligand pairing may include proteinreceptor (membranous), and its natural ligand (associated, or otherproteins or small molecules). Receptor/ligand pairs may also includeantibody/antigen, binding pairs, complementary nucleic acid, nucleicacid associating proteins and their nucleic acid ligands such asaptamers and their proteins, metal chelators and metal binding proteinligands, mimic dyes and their protein ligands, organic molecules andtheir interaction, such as hydrophobic patches, on or with biomolecules,ion exchangers and their electrostatic interaction on or withbiomolecules, and the like.

As used herein, “robotics” refers to devices and procedures capable ofthe unattended processing of samples. Preferably, the robotics operateon numerous samples in parallel to maximize the number of samplesprocessed and analyzed in a given amount of time.

The preferred embodiment of the invention is described above in theDrawings and Description of Preferred Embodiments. While thesedescriptions directly describe the above embodiments, it is understoodthat those skilled in the art may conceive modifications and/orvariations to the specific embodiments shown and described herein. Anysuch modifications or variations that fall within the purview of thisdescription are intended to be included therein as well. Unlessspecifically noted, it is the intention of the inventors that the wordsand phrases in the specification and claims be given the ordinary andaccustomed meanings to those of ordinary skill in the applicable art(s).The foregoing description of a preferred embodiment and best mode of theinvention known to the applicant at the time of filing the applicationhas been presented and is intended for the purposes of illustration, anddescription. It is not intended to be exhaustive or to limit theinvention to the precise form disclosed, and many modifications andvariations are possible in the light of the above teachings. Theembodiment was chosen and described in order to best explain theprinciples of the invention and its practical application and to enableothers skilled in the art to best utilize the invention in variousembodiments and with various modifications as are suited to theparticular use contemplated.

1. An affinity microcolumn comprising a porous molecular trap having ahigh surface area material, which has high flow properties and a lowdead volume, contained within a housing and affinity reagents bound tothe surface of the high surface area material, wherein the affinityreagents are either activated or activatable.
 2. The affinitymicrocolumn of claim 1 wherein the high surface area material of theporous molecular trap comprises silica.
 3. The affinity microcolumn ofclaim 2 wherein the porous molecular trap is formed by molding.
 4. Theaffinity microcolumn of claim 2 wherein the high surface area materialof the porous molecular trap is exposed to at least one of chemicaletching and electro etching.
 5. The affinity microcolumn of claim 1wherein the affinity reagents that are bound to the surface of the highsurface area material further comprise affinity receptors bound to theaffinity reagents.
 6. The affinity microcolumn of claim 5 furthercomprising a tethering molecule that is activated or activatible andbinds the affinity receptors to the affinity reagents.
 7. The affinitymicrocolumn of claim 5 further comprising an amplification media that isactivated or activatible and is interposed between the affinity reagentsand the affinity receptors, where the amplification media allows a highdensity of affinity receptors to be bound to the affinity reagents thanin the absence of the amplification media.
 8. The affinity microcolumnof claim 5 further comprising an amplification media interposed betweenthe affinity reagents and the affinity receptors, where theamplification media allows better access by an analyte to the affinityreceptors than in the absence of the amplification media.
 9. Theaffinity microcolumn of claim 7 wherein the amplification mediacomprises at least one of a biological polymer, a non-biological organicpolymer, and an inorganic polymer.
 10. The affinity microcolumn of claim8 wherein the amplification media comprises at least one of a biologicalpolymer, a non-biological organic polymer, and an inorganic polymer. 11.The affinity microcolumn of claim 1 wherein the housing is amicropipette.
 12. The affinity microcolumn of claim 1 wherein thehousing is a manifold having more than one microchannel, the porousmolecular trap fitting into at least one of the microchannels.
 13. Theaffinity microcolumn of claim 11 wherein the housing further comprisesat least one projection that supports the porous molecular trap.
 14. Theaffinity microcolumn of claim 11 wherein the housing further comprisesat least one projection in at least one of the microchannels, theprojection supporting the porous molecular trap.
 15. The affinitymicrocolumn of claim 1 wherein the high surface area material of theporous molecular trap comprises a polymer.
 16. The affinity microcolumnof claim 15 wherein the high surface area material of the porousmolecular trap is exposed to at least one of chemical etching andelectro etching.
 17. The affinity microcolumn of claim 15 wherein theaffinity reagents that are bound to the surface of the high surface areamaterial further comprise affinity receptors bound to the affinityreagents.
 18. The affinity microcolumn of claim 15 wherein the housingis a micropipette.
 19. The affinity microcolumn of claim 18 wherein thehousing further comprises at least one projection that supports theporous molecular trap.
 20. The affinity microcolumn of claim 15 whereinthe housing is a manifold having more than one microchannel, the porousmolecular trap fitting into at least one of the microchannels.